Synthesis of boronic ester and acid compounds

ABSTRACT

The invention relates to the synthesis of boronic ester and acid compounds. More particularly, the invention provides improved synthetic processes for the large-scale production of boronic ester and acid compounds, including the peptide boronic acid proteasome inhibitor bortezomib.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/615,894, filed Sep. 14, 2012 (pending), which is adivisional of U.S. patent application Ser. No. 12/706,063, filed Feb.16, 2010, now U.S. Pat. No. 8,283,467, which is a divisional of U.S.patent application Ser. No. 11/088,667, filed Mar. 24, 2005, now U.S.Pat. No. 7,714,159, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/557,535, filed Mar. 30, 2004 (expired). Theentire contents of each of the above-referenced patent applications areincorporated herein by this reference.

BACKGROUND OF THE INVENTION Field of the Invention

This invention relates to the synthesis of boronic ester and acidcompounds. More particularly, the invention relates to large-scalesynthetic processes for the preparation of boronic ester and acidcompounds by Lewis acid promoted rearrangement of boron “ate” complexes.

Background of the Invention

Boronic acid and ester compounds display a variety of pharmaceuticallyuseful biological activities. Shenvi et al., U.S. Pat. No. 4,499,082(1985), discloses that peptide boronic acids are inhibitors of certainproteolytic enzymes. Kettner and Shenvi, U.S. Pat. No. 5,187,157 (1993),U.S. Pat. No. 5,242,904 (1993), and U.S. Pat. No. 5,250,720 (1993),describe a class of peptide boronic acids that inhibit trypsin-likeproteases. Kleeman et al., U.S. Pat. No. 5,169,841 (1992), disclosesN-terminally modified peptide boronic acids that inhibit the action ofrenin. Kinder et al., U.S. Pat. No. 5,106,948 (1992), discloses thatcertain tripeptide boronic acid compounds inhibit the growth of cancercells.

More recently, boronic acid and ester compounds have displayedparticular promise as inhibitors of the proteasome, a multicatalyticprotease responsible for the majority of intracellular protein turnover.Ciechanover, Cell, 79: 13-21 (1994), discloses that the proteasome isthe proteolytic component of the ubiquitin-proteasome pathway, in whichproteins are targeted for degradation by conjugation to multiplemolecules of ubiquitin. Ciechanover also discloses that theubiquitin-proteasome pathway plays a key role in a variety of importantphysiological processes.

Adams et al., U.S. Pat. No. 5,780,454 (1998), U.S. Pat. No. 6,066,730(2000), U.S. Pat. No. 6,083,903 (2000), U.S. Pat. No. 6,297,217 (2001),U.S. Pat. Nos. 6,548,668, and 6,617,317 (2003), hereby incorporated byreference in their entirety, describe peptide boronic ester and acidcompounds useful as proteasome inhibitors. The references also describethe use of boronic ester and acid compounds to reduce the rate of muscleprotein degradation, to reduce the activity of NF-κB in a cell, toreduce the rate of degradation of p53 protein in a cell, to inhibitcyclin degradation in a cell, to inhibit the growth of a cancer cell, toinhibit antigen presentation in a cell, to inhibit NF-κB dependent celladhesion, and to inhibit HIV replication.

Albanell and Adams, Drugs of the Future 27: 1079-1092 (2002), disclosesthat one such peptide boronic acid proteasome inhibitor, bortezomib(N-2-pyrazinecarbonyl-L-phenylalanine-L-leucineboronic acid), showssignificant antitumor activity in human tumor xenograft models and isundergoing clinical evaluation. Richardson et al., New Engl. J. Med.,348:2609 (2003), reports the results of a Phase 2 study of bortezomib,showing its effectiveness in treating relapsed and refractory multiplemyeloma.

Studies of boronic acid protease inhibitors have been greatly advancedby the development of chemistry for the preparation of functionalizedboronic acid compounds, particularly alpha-halo- and alpha-aminoboronicacids. Matteson and Majumdar, J. Am. Chem. Soc., 102:7590 (1980),discloses a method for preparing alpha-chloroboronic esters byhomologation of boronic esters, and Matteson and Ray, J. Am. Chem. Soc.,102:7591 (1980), reports that chiral control of the homologationreaction can be achieved by the use of pinanediol boronic esters. Thepreparation of alpha-aminoboronic acid and ester compounds from thecorresponding alpha-chloroboronic esters has also been reported(Matteson et al., J. Am. Chem. Soc., 103:5241 (1981); Shenvi, U.S. Pat.No. 4,537,773 (1985)).

Matteson and Sadhu, U.S. Pat. No. 4,525,309 (1985), describes animproved procedure for the homologation of boronic esters byrearrangement of the intermediate boron “ate” complex in the presence ofa Lewis acid catalyst. The Lewis acid is reported to promote therearrangement reaction and to minimize epimerization at the alpha-carbonatom. Rigorous exclusion of water and careful control of Lewis acidstoichiometry are required for optimum results, however. These featuresrender the reaction difficult to perform successfully on a productionscale, and limit the availability of pharmaceutically important boronicester and acid compounds, such as bortezomib. Thus, there remains a needin the art for improved methods for the large-scale production ofboronic ester and acid compounds.

DESCRIPTION OF THE INVENTION

The present invention provides improved synthetic processes for thelarge-scale production of boronic ester and acid compounds. Theseprocesses offer increased yield and purity, increased throughput, andgreater ease of handling as compared to prior art methods. Notably, theprocesses described herein are suitable for batch production on a large,multi-kilogram scale that is limited only by the size of the availablemanufacturing capabilities. The processes of the invention areparticularly advantageous for the synthesis of chiral boronic ester andacid compounds, including alpha-aminoboronic ester and acid compounds.Regardless of scale, the desired products are produced with very highchemical and stereochemical purity.

The patent and scientific literature referred to herein establishesknowledge that is available to those with skill in the art. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention relates. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. The issued patents, applications, andreferences that are cited herein are hereby incorporated by reference tothe same extent as if each was specifically and individually indicatedto be incorporated by reference. In the case of inconsistencies, thepresent disclosure, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be limiting.

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%.

The term “comprises” is used herein to mean “includes, but is notlimited to.”

The term “aliphatic”, as used herein, means a straight-chain, branchedor cyclic C₁₋₁₂ hydrocarbon which is completely saturated or whichcontains one or more units of unsaturation, but which is not aromatic.For example, suitable aliphatic groups include substituted orunsubstituted linear, branched or cyclic alkyl, alkenyl, alkynyl groupsand hybrids thereof, such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or(cycloalkyl)alkenyl. In various embodiments, the aliphatic group has1-12, 1-8, 1-6, or 1-4 carbons.

The terms “alkyl”, “alkenyl”, and “alkynyl”, used alone or as part of alarger moiety, refer to a straight and branched chain aliphatic grouphaving from 1 to 12 carbon atoms, which is optionally substituted withone, two or three substituents. For purposes of the present invention,the term “alkyl” will be used when the carbon atom attaching thealiphatic group to the rest of the molecule is a saturated carbon atom.However, an alkyl group may include unsaturation at other carbon atoms.Thus, alkyl groups include, without limitation, methyl, ethyl, propyl,allyl, propargyl, butyl, pentyl, and hexyl.

For purposes of the present invention, the term “alkenyl” will be usedwhen the carbon atom attaching the aliphatic group to the rest of themolecule forms part of a carbon-carbon double bond. Alkenyl groupsinclude, without limitation, vinyl, 1-propenyl, 1-butenyl, 1-pentenyl,and 1-hexenyl. For purposes of the present invention, the term “alkynyl”will be used when the carbon atom attaching the aliphatic group to therest of the molecule forms part of a carbon-carbon triple bond. Alkynylgroups include, without limitation, ethynyl, 1-propynyl, 1-butynyl,1-pentynyl, and 1-hexynyl.

The terms “cycloalkyl”, “carbocycle”, “carbocyclyl”, “carbocyclo”, or“carbocyclic”, used alone or as part of a larger moiety, means asaturated or partially unsaturated cyclic aliphatic ring system havingfrom 3 to about 14 members, wherein the aliphatic ring system isoptionally substituted. Cycloalkyl groups include, without limitation,cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl,cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, andcyclooctadienyl. In some embodiments, the cycloalkyl has 3-6 carbons.The terms “cycloalkyl”, “carbocycle”, “carbocyclyl”, “carbocyclo”, or“carbocyclic” also include aliphatic rings that are fused to one or morearomatic or nonaromatic rings, such as decahydronaphthyl ortetrahydronaphthyl, where the radical or point of attachment is on thealiphatic ring.

The terms “haloalkyl”, “haloalkenyl” and “haloalkoxy” refer to an alkyl,alkenyl or alkoxy group, as the case may be, substituted with one ormore halogen atoms. As used herein, the term “halogen” or “halo” meansF, C, Br, or I. Unless otherwise indicated, the terms “alkyl”,“alkenyl”, and “alkoxy” include haloalkyl, haloalkenyl and haloalkoxygroups, including, in particular, those with 1-5 fluorine atoms.

The terms “aryl” and “ar-”, used alone or as part of a larger moiety,e.g., “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refer to a C₆₋₁₄aromatic moiety comprising one to three aromatic rings, which areoptionally substituted. Preferably, the aryl group is a C₆₋₁₀ arylgroup. Aryl groups include, without limitation, phenyl, naphthyl, andanthracenyl. The term “aryl”, as used herein, also includes groups inwhich an aromatic ring is fused to one or more non-aromatic rings, suchas indanyl, phenanthridinyl, or tetrahydronaphthyl, where the radical orpoint of attachment is on the aromatic ring. The term “aryl” may be usedinterchangeably with the term “aryl ring”.

An “aralkyl” or “arylalkyl” group comprises an aryl group covalentlyattached to an alkyl group, either of which independently is optionallysubstituted. Preferably, the aralkyl group is C₆₋₁₀ aryl(C₁₋₆)alkyl,including, without limitation, benzyl, phenethyl, and naphthylmethyl.

The terms “heteroaryl” and “heteroar-”, used alone or as part of alarger moiety, e.g., heteroaralkyl, or “heteroaralkoxy”, refer to groupshaving 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; having6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to four heteroatoms selected from thegroup consisting of N, O, and S. Heteroaryl groups include, withoutlimitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl,triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl,isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl,pyrazinyl, indolyl, isoindolyl, benzothienyl, benzofuranyl,dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, purinyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinoxalinyl,naphthyridinyl, pteridinyl, carbazolyl, acridinyl, and phenazinyl. Theterms “heteroaryl” and “heteroar-”, as used herein, also include groupsin which a heteroaromatic ring is fused to one or more nonaromaticrings, where the radical or point of attachment is on the heteroaromaticring. Nonlimiting examples include tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[3,4-d]pyrimidinyl. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”, any of which terms include rings that areoptionally substituted. The term “heteroaralkyl” refers to an alkylgroup substituted by a heteroaryl, wherein the alkyl and heteroarylportions independently are optionally substituted.

As used herein, the terms “heterocycle”, “heterocyclyl”, or“heterocyclic radical” refer to a stable 5- to 7-membered monocyclic or7- to 10-membered bicyclic heterocyclic moiety that is either saturatedor partially unsaturated, and having, in addition to carbon atoms, oneor more, preferably one to four, heteroatoms selected from the groupconsisting of N, O, and S, wherein the nitrogen and sulfur heteroatomsare optionally oxidized and the nitrogen atoms are optionallyquaternized. The heterocyclic ring can be attached to its pendant groupat any heteroatom or carbon atom that results in a stable structure, andany of the ring atoms can be optionally substituted. Examples of suchsaturated or partially unsaturated heterocyclic radicals include,without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl,pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl,dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, andmorpholinyl. The terms “heterocycle”, “heterocyclyl”, and “heterocyclicradical”, as used herein, also include groups in which a non-aromaticheteroatom-containing ring is fused to one or more aromatic ornon-aromatic rings, such as indolinyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on thenon-aromatic heteroatom-containing ring. The term “heterocyclylalkyl”refers to an alkyl group substituted by a heterocyclyl, wherein thealkyl and heterocyclyl portions independently are optionallysubstituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond between ring atoms. Theterm “partially unsaturated” is intended to encompass rings having oneor multiple sites of unsaturation, but is not intended to include arylor heteroaryl moieties, as herein defined.

The term “substituted”, as used herein, means that one or more hydrogenatoms of the designated moiety are replaced, provided that thesubstitution results in a stable or chemically feasible compound. Astable compound or chemically feasible compound is one in which thechemical structure is not substantially altered when kept at atemperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week, or a compound whichmaintains its integrity long enough to be useful for the syntheticprocesses of the invention. The phrase “one or more substituents”, asused herein, refers to a number of substituents that equals from one tothe maximum number of substituents possible based on the number ofavailable bonding sites, provided that the above conditions of stabilityand chemical feasibility are met.

An aryl (including the aryl moiety in aralkyl, aralkoxy, aryloxyalkyland the like) or heteroaryl (including the heteroaryl moiety inheteroaralkyl and heteroarylalkoxy and the like) group may contain oneor more substituents. Examples of suitable substituents on theunsaturated carbon atom of an aryl or heteroaryl group include -halo,—NO₂, —CN, —R*, —OR*, —SR^(o), —N(R⁺)₂, —NR⁺C(O)R*, —NR⁺C(O)N(R⁺)₂,—NR⁺CO₂R^(o), —O—CO₂R*, —O—C(O)R*, —CO₂R*, —C(O)R*, —C(O)N(R⁺)₂,—OC(O)N(R⁺)₂, —S(O)₂R^(o), —SO₂N(R⁺)₂, —S(O)R^(o), and —NR⁺SO₂N(R⁺)₂.Each R⁺ independently is selected from the group consisting of R*,—C(O)R*, —CO₂R*, and —SO₂R*, or two R⁺ on the same nitrogen atom, takentogether with the nitrogen atom, form a 5-8 membered aromatic ornon-aromatic ring having, in addition to the nitrogen, 0-2 ringheteroatoms selected from N, O, and S. Each R* independently is hydrogenor an optionally substituted aliphatic, aryl, heteroaryl, orheterocyclyl group. Each R^(o) independently is an optionallysubstituted aliphatic or aryl group.

An aliphatic group also may be substituted with one or moresubstituents. Examples of suitable substituents on the saturated carbonof an aliphatic group or of a non-aromatic heterocyclic ring include,without limitation, those listed above for the unsaturated carbon of anaryl or heteroaryl group.

The present inventors have discovered that the requirement forscrupulously dry equipment, solvents, and reagents that characterizedpreviously described procedures for the Lewis acid promotedrearrangement of boron “ate” complexes can be obviated by use of anether solvent that has low miscibility with water. Remarkably, use ofsuch a solvent permits the reaction to be run on a multi-kilogram scalewithout deterioration in yield or purity. In essence, the scale of thereaction is limited only by the size of the available manufacturingcapabilities.

In one aspect, therefore, the invention provides a large-scale processfor preparing a boronic ester compound of formula (I):

wherein:

-   -   R¹ is an optionally substituted aliphatic, aromatic, or        heteroaromatic group;    -   R² is hydrogen, a nucleofugic group, or an optionally        substituted aliphatic, aromatic, or heteroaromatic group;    -   R³ is a nucleofugic group or an optionally substituted        aliphatic, aromatic, or heteroaromatic group; and    -   each of R⁴ and R⁵, independently, is an optionally substituted        aliphatic, aromatic, or heteroaromatic group, or R⁴ and R⁵,        taken together with the intervening oxygen and boron atoms, form        an optionally substituted 5- to 10-membered ring having 0-2        additional ring heteroatoms selected from N, O, or S.

The process comprises the steps:

-   -   (a) providing a boron “ate” complex of formula (II):

where

-   -   Y is a nucleofugic group;    -   M⁺ is a cation; and    -   each of R¹ to R⁵ is as defined above; and    -   (b) contacting the boron “ate” complex of formula (II) with a        Lewis acid under conditions that afford the boronic ester        compound of formula (I), said contacting step being conducted in        a reaction mixture comprising:        -   (i) a coordinating ether solvent that has low miscibility            with water; or        -   (ii) an ether solvent that has low miscibility with water            and a coordinating co-solvent.

The previously reported processes for Lewis acid promoted rearrangementof boron “ate” complexes employ tetrahydrofuran, an ether solvent thatis fully miscible with water. Failure to employ rigorously driedequipment, solvents, and reagents in these processes results in adramatic reduction in the diastereomeric ratio. The hygroscopic Lewisacids, in particular, typically must be flame-dried immediately prior touse in the reaction. Although techniques for running moisture-sensitivereactions are familiar to those of skill in the art and are routinelypracticed on a laboratory scale, such reactions are costly and difficultto scale up.

Moreover, attempted scale-up of the prior art process frequently resultsin a further deterioration in diastereomeric ratio during workup andisolation of the product boronic ester compound. Matteson and Erdiik,Organometallics, 2:1083 (1983), reports that exposure ofalpha-haloboronic ester products to free halide ion results inepimerization at the alpha-carbon center. Without wishing to be bound bytheory, the present inventors believe that epimerization is particularlyproblematic during reaction work-up and/or subsequent steps. Forexample, epimerization is believed to occur during concentration of thereaction mixture to remove the tetrahydrofuran solvent and exchange itfor a water-immiscible solvent. Failure to completely remove thetetrahydrofuran also negatively impacts diastereomeric ratio during thesubsequent aqueous washes. Exposure of the product to halide ion duringthese steps is difficult to avoid, particularly when the reaction isperformed on a large scale.

The present inventors have discovered that the rearrangement of boron“ate” complexes is advantageously performed in an ether solvent that haslow miscibility with water. Use of such solvents obviates the need forsolvent exchange prior to the aqueous washes, and the organic-solubleproduct is effectively shielded from aqueous halide ion during thewashes, even if performed on a large scale. Preferably, the solubilityof water in the ether solvent is less than about 5% w/w, more preferablyless than about 2% w/w. In various embodiments, ether solvent that haslow miscibility with water constitutes at least about 70%, at leastabout 80%, at least about 85%, at least about 90%, or at least about 95%v/v of the reaction mixture.

The ether solvent preferably is one that is suitable for routine use inlarge-scale production. As used herein, the term “large-scale” refers toa reaction that utilizes at least about five moles of at least onestarting material. Preferably, a large-scale process utilizes at leastabout 10, 20, 50, or 100 moles of at least one starting material.

For purposes of the invention, the term “ether” refers to any of a classof chemical compounds characterized in having an oxygen atom attached totwo carbon atoms. An “ether solvent” is an ether compound that exists inliquid form at the desired reaction temperature and is capable ofdissolving the starting material(s) and/or product(s) of the reaction.Non-limiting examples of ether solvents suitable for use in the processof the invention include tert-butyl methyl ether, tert-butyl ethylether, tert-amyl methyl ether, and isopropyl ether.

In one embodiment, the reaction mixture further comprises a coordinatingco-solvent. In another embodiment, the ether solvent that has lowmiscibility with water is sufficiently coordinating that a coordinatingco-solvent is not necessary. For purposes of the invention, the terms“coordinating co-solvent” and “coordinating solvent” refer to a solventthat is capable of coordinating the Lewis acid and solvating the ioniccomponents of the reaction. Hindered ether solvents, such as tert-butylmethyl ether, are poorly coordinating and preferably are used with acoordinating co-solvent. Nonlimiting examples of coordinatingco-solvents suitable for use in the practice of the invention includetetrahydrofuran, dioxane, water, and mixtures thereof.

In some embodiments, the reaction mixture comprises at least about 5% orat least about 10% v/v of a coordinating co-solvent. Preferably, theamount of a water-miscible coordinating co-solvent present in thereaction mixture is not so great as to interfere with phase separationduring the reaction or workup. In various embodiments, the coordinatingco-solvent constitutes no more than about 20%, about 15%, or about 10%v/v of the reaction mixture.

As used herein, the term “nucleofugic” refers to any group that iscapable of undergoing nucleophilic displacement under the rearrangementconditions of the present process. Such nucleofugic groups are known inthe art. Preferably, the nucleofugic group is a halogen, more preferablychloro or bromo. In the course of the rearrangement reaction convertingthe boron “ate” complex of formula (II) into the boronic ester compoundof formula (I), the nucleofugic group Y is released as Y⁻. By way ofexample, when Y is chloro, chloride ion is released in step (b).

The variable M⁺ is any cationic counterion for the negatively chargedtetravalent boron atom in the boron “ate” complex of formula (II). Insome preferred embodiments, M⁺ is selected from the group consisting ofLi⁺, Na⁺, and K⁺. One of skill in the art will recognize that the saltM⁺Y⁻ is formed as a byproduct in the rearrangement reaction of step (b).

The variable R¹ preferably is a group with good migratory aptitude. Insome embodiments, R¹ is C₁₋₈ aliphatic, C₆₋₁₀ aryl, or (C₆₋₁₀ aryl)(C₁₋₆aliphatic), any of which groups is optionally substituted. In certainembodiments, R¹ is C₁₋₄ aliphatic, particularly isobutyl.

The variable R² preferably is hydrogen, a nucleofugic group, or anoptionally substituted C_(1-s) aliphatic, C₆₋₁₀ aryl, or (C₆₋₁₀aryl)(C₁₋₆ aliphatic) group. The variable R³ preferably is a nucleofugicgroup or an optionally substituted C_(1-s) aliphatic, C₆₋₁₀ aryl, or(C₆₋₁₀ aryl)(C₁₋₆ aliphatic) group. One of skill in the art willrecognize that functional substituents may be present on any of R¹, R²,or R³, provided that the functional substituent does not interfere withthe formation of the boron “ate” complex of formula (II).

One embodiment of the invention relates to a process for preparing aboronic ester compound of formula (I), wherein R³ is a nucleofugicgroup. Such compounds are useful as intermediates for the synthesis ofalpha-substituted boronic ester and acid compounds, includingalpha-aminoboronic ester and acid compounds, as described below. Incertain preferred embodiments, R³ is a nucleofugic group and R² ishydrogen.

The variables R⁴ and R⁵ can be the same or different. In someembodiments, R⁴ and R are directly linked, so that R⁴ and R⁵, takentogether with the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring, which can have 0-2 additional ringheteroatoms selected from N, O, or S. In some embodiments, the ring is a5- or 6-membered ring, preferably a 5-membered ring.

The present invention is particularly advantageous for the Lewis acidpromoted rearrangement of boron “ate” complexes of formula (II), whereinR⁴ and R⁵ are directly linked and together are a chiral moiety. Oneembodiment of the invention relates to the rearrangement of such chiralboron “ate” complexes to provide a boronic ester compound of formula (I)wherein the carbon atom bearing R¹, R², and R³ is a chiral center. Therearrangement reaction preferably proceeds with a high degree ofstereodirection by the R⁴-R⁵ chiral moiety to provide the boronic estercompound of formula (I) having a diastereomeric ratio at the carbon atombearing R¹, R², and R³ of at least about 96:4 relative to a chiralcenter in the R⁴-R⁵ chiral moiety. Preferably, the diastereomeric ratiois at least about 97:3.

The terms “stereoisomer”, “enantiomer”, “diastereomer”, “epimer”, and“chiral center”, are used herein in accordance with the meaning each isgiven in ordinary usage by those of ordinary skill in the art. Thus,stereoisomers are compounds that have the same atomic connectivity, butdiffer in the spatial arrangement of the atoms. Enantiomers arestereoisomers that have a mirror image relationship, that is, thestereochemical configuration at all corresponding chiral centers isopposite. Diastereomers are stereoisomers having more than one chiralcenter, which differ from one another in that the stereochemicalconfiguration of at least one, but not all, of the corresponding chiralcenters is opposite. Epimers are diastereomers that differ instereochemical configuration at only one chiral center.

As used herein, the term “diastereomeric ratio” refers to the ratiobetween diastereomers which differ in the stereochemical configurationat one chiral center, relative to a second chiral center in the samemolecule. By way of example, a chemical structure with two chiralcenters provides four possible stereoisomers: R*R, R*S, S*R, and S*S,wherein the asterisk denotes the corresponding chiral center in eachstereoisomer. The diastereomeric ratio for such a mixture ofstereoisomers is the ratio of one diastereomer and its enantiomer to theother diastereomer and its enantiomer=(R*R+S*S):(R*S+S*R).

One of ordinary skill in the art will recognize that additionalstereoisomers are possible when the molecule has more than two chiralcenters. For purposes of the present invention, the term “diastereomericratio” has identical meaning in reference to compounds with multiplechiral centers as it does in reference to compounds having two chiralcenters. Thus, the term “diastereomeric ratio” refers to the ratio ofall compounds having R*R or S*S configuration at the specified chiralcenters to all compounds having R*S or S*R configuration at thespecified chiral centers. For convenience, this ratio is referred toherein as the diastereomeric ratio at the asterisked carbon, relative tothe second specified chiral center.

The diastereomeric ratio can be measured by any analytical methodsuitable for distinguishing between diastereomeric compounds havingdifferent relative stereochemical configurations at the specified chiralcenters. Such methods include, without limitation, nuclear magneticresonance (NMR), gas chromatography (GC), and high performance liquidchromatography (HPLC) methods.

As discussed above, one embodiment of the invention is directed toprocesses that provide a boronic ester compound of formula (I) having adiastereomeric ratio at the carbon atom bearing R¹, R², and R³ of atleast about 96:4 relative to a chiral center in the R⁴-R⁵ chiral moiety.One of skill in the art will recognize that the R⁴-R⁵ chiral moiety mayitself contain more than one chiral center. When R⁴-R⁵ does have morethan one chiral center, it preferably has high diastereomeric purity,and the diastereomeric ratio at the carbon atom bearing R¹, R², and R³can be measured relative to any one of the chiral centers in R⁴-R⁵.

In the processes of the invention, the R⁴-R⁵ chiral moiety preferablyhas a high level of enantiomeric purity. For purposes of the invention,the term “enantiomeric purity” is used to mean “enantiomeric excess”,which is the amount by which the major enantiomer is in excess of theminor enantiomer, expressed as a percentage of the total. Preferably,the R⁴-R⁵ chiral moiety has an enantiomeric purity of at least about98%, more preferably at least about 99%, still more preferably at leastabout 99.5%, and most preferably at least about 99.9%.

When the R⁴-R⁵ chiral moiety has very high enantiomeric purity, thediastereomeric ratio at the carbon atom bearing R¹, R², R³ approximatesthe epimeric ratio at that center, i.e., diastereomericratio=(R*R):(S*R) or (R*S):(S*S)≅(R*):(S*). As used herein, the term“epimeric ratio” refers to the ratio of product having one absolutestereochemical configuration at a given chiral center to product havingthe opposite absolute stereochemical configuration at the correspondingchiral center. Preferably, the products have identical stereochemicalconfiguration at all other corresponding chiral centers. In oneembodiment, therefore, the invention relates to the rearrangement of achiral boron “ate” complex of formula (II) to provide a boronic estercompound of formula (I) wherein the epimeric ratio at the carbon atombearing R¹, R², and R³ is at least about 96:4, more preferably at leastabout 97:3.

Lewis acids suitable for use in the practice of the invention are thosecapable of complexing with the nucleofugic group to facilitate itsdisplacement upon migration of R¹. Preferably, the Lewis acid isadditionally capable of coordinating with an oxygen atom attached toboron. Nonlimiting examples of suitable Lewis acids include zincbromide, zinc chloride, ferric bromide, and ferric chloride. In certainpreferred embodiments, the Lewis acid is zinc chloride.

The contacting step preferably is performed at low temperature, but maybe performed at ambient or elevated temperature. The selection of anappropriate reaction temperature will depend largely on the Lewis acidemployed, as well as the migratory aptitude of the R¹ moiety. Oneskilled in the art will be able to select a suitable temperature in viewof the reaction conditions being used.

In some embodiments, the contacting step is performed at a reactiontemperature of at least about −100° C., −78° C., or −60° C. In someembodiments, the contacting step is performed at a reaction temperaturethat is no greater than about 80° C., 40° C., or 30° C. Any rangeencompassing these high and low temperatures are included within thescope of the invention. Preferably, the contacting step is performed ata reaction temperature in the range of about −100° C. to about 80° C.,about −70° C. to about 40° C., about −60° C. to about 30° C., or about−50° C. to about 30° C. In certain preferred embodiments, the contactingstep is begun at low temperature, preferably in the range of about −70°C. to about −30° C., and then the reaction mixture is allowed to warm,preferably to ambient temperature.

Surprisingly, the process of the present invention requires no specialprecautions to avoid the presence of water during the rearrangementreaction itself. In some embodiments, moist Lewis acid is employed, withminimal deterioration in diastereomeric ratio. When used in reference tothe Lewis acid, the term “moist” means that the water content of theLewis acid is greater than about 100, 200, 500, or 1,000 ppm.Remarkably, the Lewis acid even can be added to the reaction mixture inthe form of an aqueous solution without deleterious impact ondiastereomeric ratio.

In some embodiments, therefore, the process of the invention comprisesthe steps:

-   -   (a) providing a solution comprising a boron “ate” complex of        formula (II) and        -   (i) a coordinating ether solvent that has low miscibility            with water; or        -   (ii) an ether solvent that has low miscibility with water            and a coordinating co-solvent; and    -   (b) adding to the solution of step (a) a Lewis acid solution        comprising water and a Lewis acid.

In some other embodiments, the Lewis acid solution comprisestetrahydrofuran and a Lewis acid.

Thus, unlike the prior art process, the process of the invention isreadily amenable to large-scale production. In various embodiments, atleast about 5, 10, 20, 50, 100, 500, or 1000 moles of boron “ate”complex of formula (II) is contacted with a Lewis acid under conditionsthat afford the boronic ester compound of formula (I). The inventionfurther provides a composition comprising a boronic ester compound offormula (I), as described herein, and an ether solvent that has lowmiscibility with water. The composition preferably comprises at leastabout 5, 10, 20, 50, 100, 500, or 1000 moles of the boronic estercompound of formula (I). In certain embodiments, R⁴ and R⁵ together area chiral moiety, and the compound of formula (I) present in thecomposition has a diastereomeric ratio of at least about 96:4 at thecarbon atom bearing R¹, R², and R³, relative to a chiral center in theR⁴-R⁵ chiral moiety.

Workup of the reaction preferably comprises washing the reaction mixturewith an aqueous solution and concentrating the washed reaction mixtureby removal of solvents to afford a residue comprising the boronic estercompound of formula (I). Preferably, the residue comprises at leastabout 5, 10, 20, 50, 100, 500, or 1000 moles of the boronic estercompound of formula (I). In those embodiments wherein R⁴-R⁵ is a chiralmoiety, the boronic ester compound of formula (I) present in the residuepreferably has a diastereomeric ratio of at least about 96:4 at thecarbon atom bearing R¹, R², and R³, relative to a chiral center in theR⁴-R⁵ chiral moiety. More preferably, the diastereomeric ratio is atleast about 97:3.

The boron “ate” complex of formula (II) can be prepared by any knownmethod, but preferably is prepared by reaction of a boronic ester offormula (III):

with a reagent of formula (IV):

wherein each of M⁺, Y, and R¹ to R⁵ are as defined above for the boron“ate” complex of formula (II). In some embodiments, the reaction isperformed at a reaction temperature of at least about −100° C., −78° C.or −60° C. In some embodiments, the reaction is performed at a reactiontemperature no greater than about 0° C., −20° C. or −40° C. Any rangeencompassing these high and low temperatures are included within thescope of the invention. The reaction preferably is performed at areaction temperature in the range of about −100° C. to about 0° C.,about −78° C. to about −20° C., or about −60° C. to about −40° C. Insome embodiments, the boron “ate” complex of formula (II) is prepared ina solution comprising an ether solvent having low miscibility withwater, and the reaction mixture is directly treated with a Lewis acid toeffect rearrangement to the boron ester compound of formula (I).

In some embodiments, the reagent of formula (IV) is formed in situ. Suchembodiments include the steps:

-   -   (i) providing a solution comprising a boronic ester of formula        (III), as defined above, and a compound of formula (V):

wherein R² and R³ are as defined above for the reagent of formula (IV);and

-   -   (ii) treating the solution with a strong, sterically hindered        base to form the boron “ate” complex of formula (II).

In some embodiments, the sterically hindered base is an alkali metaldialkylamide bases of formula M²N(R^(#))₂, where M² is Li, Na, or K, andeach R^(#), independently is a branched or cyclic C₃₋₆ aliphatic. Insitu formation of the reagent of formula (IV) is especially advantageousin those embodiments wherein Y is a nucleofugic group, due to theinstability of the reagent of formula (IV).

The boronic ester of formula (III) can be prepared by any known method,but typically is prepared by esterification of the corresponding boronicacid compound, e.g., by methods described in Brown et al.,Organometallics, 2: 1311-1316 (1983). Cyclic boronic esters of formula(III) preferably are prepared by:

-   -   (a) providing a solution comprising:        -   (i) a boronic acid compound of formula R¹—B(OH)₂;        -   (ii) a compound of formula HO—R⁴-R⁵—OH, wherein R⁴ and R⁵,            taken together, are an optionally substituted linking chain            comprising 2-5 carbon atoms and 0-2 heteroatoms selected            from the group consisting of O, N, and S; and (iii) an            organic solvent that forms an azeotrope with water; and    -   (b) heating the solution at reflux with azeotropic removal of        water.

As used in reference to R⁴ and R⁵, the term “linking chain” refers tothe shortest linear chain of atoms connecting the oxygen atoms to whichR⁴ and R⁵ are attached. The linking chain optionally is substituted atany chain atom, and one or more chain atoms also may form part of a ringsystem that is spiro to, fused to, or bridging the linear linking chain.By way of example, but not limitation, in some embodiments, the compoundof formula HO—R⁴-R⁵—OH is pinanediol, having the structure:

In such embodiments, the linking chain R⁴-R⁵ comprises two carbon atoms,which together form one side of the bicyclo[3.1.1]heptane ring system,and one of which additionally is substituted with a methyl group.

In some embodiments, the compound of formula HO—R⁴-R⁵—OH is a chiraldiol, preferably one having high diastereomeric and enantiomeric purity.One of skill in the art will appreciate that in such embodiment, thecompound of formula HO—R⁴-R⁵—OH is employed as a chiral auxiliary todirect the stereochemical configuration at the carbon bearing R¹, R²,and R³. Chiral diols useful as chiral auxiliaries in organic synthesisare well-known in the art. Nonlimiting examples include 2,3-butanediol,preferably (2R,3R)-(−)-2,3-butanediol or (2S,3S)-(+)-2,3-butanediol;pinanediol, preferably (1R,2R,3R,5S)-(−)-pinanediol or(1S,2S,3S,5R)-(+)-pinanediol; 1,2-cyclopentanediol, preferably(1S,2S)-(+)-trans-1,2-cyclopentanediol or(1R,2R)-(−)-trans-1,2-cyclopentanediol; 2,5-hexanediol, preferably(2S,5S)-2,5-hexanediol or (2R,5R)-2,5-hexanediol;1,2-dicyclohexyl-1,2-ethanediol, preferably(1R,2R)-1,2-dicyclohexyl-1,2-ethanediol or(1S,2S)-1,2-dicyclohexyl-1,2-ethanediol; hydrobenzoin, preferably(S,S)-(−)-hydrobenzoin or (R,R)-(+)-hydrobenzoin; 2,4-pentanediol,preferably (R,R)-(−)-2,4-pentanediol or (S,S,)-(+)-2,4-pentanediol;erythronic γ-lactone, preferably D-erythronic γ-lactone. Carbohydrates,e.g. a 1,2,5,6-symmetrically protected mannitol, also may be used aschiral diols.

Nonlimiting examples of organic solvents suitable for use in theesterification reaction include acetonitrile, toluene, hexane, heptane,and mixtures thereof. In some embodiments, the organic solvent is anether solvent, preferably an ether solvent that has low miscibility withwater. In certain preferred embodiments, the esterification reaction isperformed in an ether solvent that has low miscibility with water, andthe product solution comprising the boronic ester of formula (III) isused directly in the next step, without isolation of the boronic ester.

As noted above, the process of the present invention for the first timepermits workup of large-scale reactions without significantdeterioration in diastereomeric ratio. In another aspect, therefore, theinvention provides a composition comprising at least about 5, 10, 20,50, 100, 500, or 1000 moles of a boronic ester compound of formula (I):

wherein:

-   -   R¹ is an optionally substituted aliphatic, aromatic, or        heteroaromatic group;    -   R² is hydrogen, a nucleofugic group, or an optionally        substituted aliphatic, aromatic, or heteroaromatic group;    -   R³ is a nucleofugic group or an optionally substituted        aliphatic, aromatic, or heteroaromatic group; and    -   R⁴ and R⁵, taken together with the intervening oxygen and boron        atoms, form an optionally substituted 5- to 10-membered chiral        ring having 0-2 additional ring heteroatoms selected from N, O,        or S;    -   wherein the carbon atom to which R¹, R², and R³ are attached is        a chiral center, having a diastereomeric ratio of at least about        96:4, preferably at least about 97:3, relative to a chiral        center in the R⁴-R⁵ chiral moiety.

Preferred values for R¹ to R³ are as described above. Preferably,solvents constitute less than about 30% w/w, 20% w/w, 10% w/w, or 5% w/wof the composition according to this aspect of the invention. In someembodiments, the boronic ester compound of formula (I) constitutes atleast about 70% w/w, 80% w/w, 90% w/w, or 95% w/w of the composition.

One embodiment relates to the composition described above, wherein atleast one of the following features is present:

-   -   (a) R³ is chloro;    -   (b) the boronic ester compound (I) is

-   -   (c) R² is hydrogen; and    -   (d) R¹ is C₁₋₄ aliphatic.

All of the boronic ester compound of formula (I) present in thecomposition may be produced in a single batch run. For purposes of theinvention, the term “batch run” refers to execution of a syntheticprocess, wherein each step of the process is performed only once.Preferably, the boronic ester compound of formula (I) present in thecomposition is prepared in a single batch run of the process accordingto the first aspect of the invention. One of ordinary skill in the artwill appreciate that preparation of a given quantity of product by asingle batch run of a large-scale process is more efficient and providesa more homogeneous product than preparation of the same quantity ofproduct by repeated execution of a small-scale process.

The boronic ester compounds of formula (I) wherein R³ is a nucleofugicgroup are useful as intermediates for the synthesis ofalpha-aminoboronic ester compounds. In another aspect, therefore, theinvention provides a large-scale process for preparing analpha-aminoboronic ester, preferably by a process comprising the steps:

-   -   (a) providing a boron “ate” complex of formula (II):

where

-   -   Y is a nucleofugic group;    -   M⁺ is a cation;    -   R¹ is an optionally substituted aliphatic, aromatic, or        heteroaromatic group;    -   R² is hydrogen;    -   R³ is a nucleofugic group; and    -   each of R⁴ and R⁵, independently, is an optionally substituted        aliphatic, aromatic, or heteroaromatic group, or R⁴ and R⁵,        taken together with the intervening oxygen and boron atoms, form        an optionally substituted 5- to 10-membered ring having 0-2        additional ring heteroatoms selected from N, O, or S;    -   (b) contacting the boron “ate” complex of formula (II) with a        Lewis acid under conditions that afford the boronic ester        compound of formula (I):

where each of R¹ to R⁵ is as defined above, said contacting step beingconducted in a reaction mixture comprising:

-   -   (i) a coordinating ether solvent that has low miscibility with        water; or    -   (ii) an ether solvent that has low miscibility with water and a        coordinating co-solvent; and    -   (c) treating the boronic ester compound of formula (I) with a        reagent of formula M¹-N(G)₂, where M¹ is an alkali metal and        each G individually or together is an amino group protecting        group to form a byproduct of formula M¹-R³ and a compound of        formula (VIII):

wherein each G and R¹ to R⁵ are as defined above; and

-   -   (d) removing the G groups to form a compound of formula (VII):

or an acid addition salt thereof.

In some embodiments, in step (c), the boronic ester compound of formula(I) is treated with a reagent of formula M¹-N(Si(R⁶)₃)₂ where M¹ is analkali metal and each R⁶ independently is selected from the groupconsisting of alkyl, aralkyl, and aryl, where the aryl or aryl portionof the aralkyl is optionally substituted.

Reaction of the boronic ester compound of formula (I) with the reagentof formula M¹-N(G)₂ preferably is conducted at a reaction temperature inthe range of about −100° C. to about 50° C., preferably about −50° C. toabout 25° C., and more preferably about −30° C. to about 0° C. In someembodiments, R³ is halo, preferably chloro, and M¹ is Li. To facilitateisolation of the product of formula (VIII), the reaction mixturepreferably comprises an organic solvent in which the byproduct M¹-R³ haslow solubility. Nonlimiting examples of suitable organic solventsinclude methylcyclohexane, cyclohexane, heptane, hexane, and toluene. Insome embodiments, step (c) further comprises filtering the reactionmixture to remove M¹-R³ and provide a filtrate comprising the compoundof formula (VIII). Preferably, the filtrate is used directly in step(d).

In those embodiments wherein the reaction mixture comprises an organicsolvent in which the byproduct M¹-R³ has low solubility, the reactionmixture may additionally comprise a solvent in which the byproduct M¹-R³has high solubility. In such cases, the solvent in which the byproductM¹-R³ has high solubility preferably is removed prior to filtration ofthe reaction mixture. By way of example, in some embodiments, a reagentof formula M¹-N(Si(R⁶)₃)₂ is added to the reaction mixture as a solutioncomprising tetrahydrofuran. In such embodiments, step (c) preferablyfurther comprises removing the tetrahydrofuran before filtering thereaction mixture.

Those of skill in the art are aware of various methods that can be usedto remove the protecting groups G in the compound of formula (VIII),including, e.g., aqueous hydrolysis or treatment with acid. The productalpha-aminoboronic ester of formula (VII) has low stability andpreferably is immediately derivatized (Matteson et al., J. Am. Chem.Soc., 103:5241 (1981)) or is isolated as an acid addition salt. In someembodiments, step (d) comprises treating the compound of formula (VIII)with an acid and isolating the compound of formula (VII) as the acidaddition salt. In certain preferred embodiments, the acid istrifluoroacetic acid, and the compound of formula (VII) is isolated asthe trifluoroacetic acid addition salt.

As discussed above, the processes of the invention are particularlywell-suited for preparing alpha-aminoboronic ester compounds of formula(VII), wherein the alpha carbon is a chiral center. Thus, one embodimentof the invention relates to a large-scale process for preparing analpha-aminoboronic ester compound of formula (VIIa) or (VIIb):

or an acid addition salt thereof, wherein:

-   -   R¹ is an optionally substituted aliphatic, aromatic, or        heteroaromatic group; and    -   R⁴ and R⁵, taken together with the intervening oxygen and boron        atoms, form an optionally substituted chiral cyclic boronic        ester;        said process comprising:    -   (a) providing a boron “ate” complex of formula (IIa) or (IIb):

where

-   -   Y is a nucleofugic group;    -   M⁺ is a cation;    -   R² is hydrogen;    -   R³ is a nucleofugic group; and    -   R⁴ and R⁵ are as defined above;    -   (b) contacting the boron “ate” complex of formula (Ha) or (IIb)        with a Lewis acid under conditions that afford a boronic ester        compound of formula (Ia) or (Ib):

where each of R¹ to R⁵ is as defined above, said contacting step beingconducted in a reaction mixture comprising:

-   -   (i) a coordinating ether solvent that has low miscibility with        water; or    -   (ii) an ether solvent that has low miscibility with water and a        coordinating co-solvent; and    -   (c) treating the boronic ester compound of formula (Ia) or (Ib)        with a reagent of formula M¹-N(G)₂, where M¹ is an alkali metal        and G is an amino group protecting moiety, to form a compound of        formula (VIIIa) or (VIIIb):

wherein each G and R¹ to R⁵ are as defined above; and

-   -   (d) removing the G groups to form a compound of formula (VIIa)        or (VIIb):

or an acid addition salt thereof.

Preferred values for Y, M⁺, R¹ to R⁵, and G are as described above. Thecompound of formula (VIIa) or (VIIb) preferably has a diastereomericratio at the alpha-carbon of at least about 96:4, more preferably atleast about 97:3, relative to a chiral center in the R⁴-R⁵ chiralmoiety.

The alpha-aminoboronic ester compounds of formula (VII) are usefulsynthetic intermediates for the preparation of peptidyl boronic estercompounds. In some embodiments, therefore, the process according to thisaspect of the invention further comprises coupling the compound offormula (VII) with a compound of formula (IX):

wherein:

-   -   P¹ is an amino group blocking moiety;    -   R⁷ is selected from the group consisting of hydrogen,        C₁₋₁₀aliphatic, optionally substituted C₆₋₁₀aryl, or        C₁₋₆aliphatic-R⁸; and    -   R⁸ is selected from the group consisting of alkoxy, alkylthio,        optionally substituted aryl, heteroaryl, and heterocyclyl        groups, and optionally protected amino, hydroxy, and guanidino        groups; and    -   X is OH or a leaving group;        to form a compound of formula (X):

wherein each of P¹, R¹, R⁴, R⁵, and R⁷ is as defined above.

The leaving group X is any group capable of nucleophilic displacement bythe alpha-amino group of the compound of formula (VII). In someembodiments, the moiety —C(O)—X is an activated ester, such as anO—(N-hydroxysucccinimide) ester. In some embodiments, an activated esteris generated in situ by contacting a compound of formula (IX), wherein Xis OH, with a peptide coupling reagent. Examples of suitable peptidecoupling reagents include, without limitation, carbodiimide reagents,e.g., dicyclohexylcarbodiimide (DCC) or1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC); phosphoniumreagents, e.g., benzotriazol-1-yloxytris(dimethylamino)phosphoniumhexafluorophosphate (BOP reagent); and uronium reagents, e.g.,O-(1H-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate(TBTU).

Those of skill in the art also are aware of procedures that permit thedirect coupling of silyl protected amines, without a prior deprotectionstep. In such procedures, the silyl groups are removed in situ under thecoupling reaction conditions. In some embodiments of the presentinvention, therefore, a compound of formula (VIII) is contacted with acompound of formula (IX) under conditions that remove the (R⁶)₃Si groupsin situ and form a compound of formula (X).

For purposes of the invention, the term “amino-group blocking moiety”refers to any group used to derivatize an amino group, especially anN-terminal amino group of a peptide or amino acid. The term “amino-groupblocking moiety” includes, but is not limited to, protecting groups thatare commonly employed in organic synthesis, especially peptidesynthesis. See, for example, Gross and Mienhoffer, eds., The Peptides,Vol. 3, Academic Press, New York, 1981, pp. 3-88; Green and Wuts,Protective Groups in Organic Synthesis, 3^(rd) edition, John Wiley andSons, Inc., New York, 1999. Unless otherwise specified, however, it isnot necessary for an amino-group blocking moiety to be readilycleavable. Amino-group blocking moieties include, e.g., alkyl, acyl,alkoxycarbonyl, aminocarbonyl, and sulfonyl moieties. In someembodiments, the amino-group blocking moiety is an acyl moiety derivedfrom an amino acid or peptide, or a derivative or analog thereof.

As used herein, the term “amino acid” includes both naturally occurringand unnatural amino acids. For purposes of the invention, a “derivative”of an amino acid or peptide is one in which a functional group, e.g., ahydroxy, amino, carboxy, or guanidino group at the N-terminus or on aside chain, is modified with a blocking group. As used herein, an“analog” of an amino acid or peptide is one which includes a modifiedbackbone or side chain. The term “peptide analog” is intended to includepeptides wherein one or more stereocenters are inverted and one or morepeptide bonds are replaced with a peptide isostere.

In some embodiments, P¹ is a cleavable protecting group. Examples ofcleavable protecting groups include, without limitation, acyl protectinggroups, e.g., formyl, acetyl (Ac), succinyl (Suc), or methoxysuccinyl(MeOSuc), and urethane protecting groups, e.g., tert-butoxycarbonyl(Boc), benzyloxycarbonyl (Cbz), or fluorenylmethoxycarbonyl (Fmoc).

In some such embodiments, the process according to this aspect of theinvention further comprises the steps:

-   -   (f) removing the protecting group P¹ to form a compound of        formula (XI):

or an acid addition salt thereof, wherein each of R¹, R⁴, R⁵, and R⁷ isas defined above; and

-   -   (g) coupling the compound of formula (XI) with a reagent of        formula P²—X, wherein P² is any amino group blocking moiety, as        described above, and X is a leaving group, to form a compound of        formula (XII):

wherein each of P², R¹, R⁴, R⁵, and R⁷ are as defined above. One ofskill in the art will recognize that in those embodiments wherein P² isan acyl group, including, e.g., an acyl moiety derived from an aminoacid or peptide, or an analog or derivative thereof, the leaving group Xmay be generated in situ, as discussed above for the compound of formula(IX).

In each of the compounds (X) and (XII), the boronic acid moiety isprotected as a boronic ester. If desired, the boronic acid moiety can bedeprotected by any method known in the art. Preferably, the boronic acidmoiety is deprotected by transesterification in a biphasic mixture. Morepreferably, the boronic acid deprotecting step comprises the steps:

-   -   (i) providing a biphasic mixture comprising the boronic ester        compound of formula (X) or (XII), an organic boronic acid        acceptor, a lower alkanol, a C₅₋₈ hydrocarbon solvent, and        aqueous mineral acid;    -   (ii) stirring the biphasic mixture to afford the corresponding        deprotected boronic acid compound of formula (Xa) or (XIII):

-   -   (iii) separating the solvent layers; and    -   (iv) extracting the compound of formula (Xa), (XIII), or a        boronic acid anhydride thereof, into an organic solvent.

The organic boronic acid acceptor in step (i) preferably is analiphatic, aryl, or ar(aliphatic)boronic acid. In some embodiments, theboronic acid acceptor is selected from the group consisting ofphenylboronic acid, benzylboronic acid, butylboronic acid, pentylboronicacid, hexylboronic acid, and cyclohexylboronic acid. In certainembodiments, the boronic acid acceptor is isobutylboronic acid. In someembodiments, the boronic acid acceptor is selected so that the boronicester compound of formula (III) is formed as a byproduct of thedeprotection reaction. The boronic ester compound of formula (III) canthen be used in another batch run of the process described above. Insuch embodiments, the moiety R⁴-R⁵ is effectively recycled, which may beparticularly advantageous if R⁴-R⁵ is an expensive chiral moiety.

To enhance the purity of the product, the aqueous layer containing thecompound of formula (Xa) or (XIII) preferably is washed to removeneutral organic impurities prior to the extracting step (iv). In suchembodiments, step (iii) preferably comprises the steps:

-   -   (1) separating the solvent layers;    -   (2) adjusting the aqueous layer to basic pH;    -   (3) washing the aqueous layer with an organic solvent; and    -   (4) adjusting the aqueous layer to a pH less than about 6.

In some embodiments, the invention relates to an improved process formanufacturing the proteasome inhibitor bortezomib. Thus, in oneembodiment, the invention provides a large-scale process for forming acompound of formula (XIV):

or a boronic acid anhydride thereof. The process comprises the steps:

-   -   (a) providing a boron “ate” complex of formula (XV):

wherein:

-   -   R³ is a nucleofugic group;    -   Y is a nucleofugic group; and    -   M⁺ is an alkali metal;    -   (b) contacting the boron “ate” complex of formula (XV) with a        Lewis acid under conditions that afford a boronic ester compound        of formula (XVI):

said contacting step being conducted in a reaction mixture comprising:

-   -   (i) a coordinating ether solvent that has low miscibility with        water; or    -   (ii) an ether solvent that has low miscibility with water and a        coordinating co-solvent;    -   (c) treating the boronic ester compound of formula (XVI) with a        reagent of formula M¹-N(G)₂, where M¹ is an alkali metal and        each G individually or together is an amino group protecting        group, to form a compound of formula (XVII):

-   -   (d) removing the G groups to form a compound of formula (XVIII):

or an acid addition salt thereof;

-   -   (e) coupling the compound of formula (XVIII) with a compound of        formula (XIX);

wherein:

-   -   P¹ is a cleavable amino group protecting moiety; and    -   X is OH or a leaving group;        to form a compound of formula (XX):

wherein P¹ is as defined above;

-   -   (f) removing the protecting group P¹ to form a compound of        formula (XXI):

or an acid addition salt thereof;

-   -   (g) coupling the compound of formula (XXI) with a reagent of        formula (XXII)

wherein X is a OH or a leaving group, to form a compound of formula(XXIII):

and

-   -   (h) deprotecting the boronic acid moiety to form the compound of        formula (XIV) or a boronic acid anhydride thereof.

In some embodiments, the process is characterized by at least one of thefollowing features (1)-(5). In certain preferred embodiments, theprocess is characterized by all five features (1)-(5) below.

-   -   (1) In the boron “ate” complex of formula (XV), R³ and Y both        are chloro.    -   (2) The coupling step (e) comprises the steps:        -   (i) coupling the compound of formula (XVIII) with a compound            of formula (XIX) wherein X is OH in the presence of            2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium            tetrafluoroborate (TBTU) and a tertiary amine in            dichloromethane;        -   (ii) performing a solvent exchange to replace            dichloromethane with ethyl acetate; and        -   (iii) performing an aqueous wash of the ethyl acetate            solution.    -   (3) The protecting group removing step (f) comprises the steps:        -   (i) treating the compound of formula (XX) with HCl in ethyl            acetate;        -   (ii) adding heptane to the reaction mixture; and        -   (iii) isolating by crystallization the compound of            formula (XXI) as its HCl addition salt.    -   (4) The coupling step (g) comprises the steps:        -   (i) coupling the compound of formula (XXI) with            2-pyrazinecarboxylic acid in the presence of TBTU and a            tertiary amine in dichloromethane;        -   (ii) performing a solvent exchange to replace            dichloromethane with ethyl acetate; and        -   (iii) performing an aqueous wash of the ethyl acetate            solution.    -   (5) The boronic acid deprotecting step (h) comprises the steps:        -   (i) providing a biphasic mixture comprising the compound of            formula (XXIII), an organic boronic acid acceptor, a lower            alkanol, a C₅₋₈ hydrocarbon solvent, and aqueous mineral            acid;        -   (ii) stirring the biphasic mixture to afford the compound of            formula (XIV);        -   (iii) separating the solvent layers; and        -   (iv) extracting the compound of formula (XIV), or a boronic            acid anhydride thereof, into an organic solvent.

Preferably, step (h)(iii) comprises the steps:

-   -   (1) separating the solvent layers;    -   (2) adjusting the aqueous layer to basic pH;    -   (3) washing the aqueous layer with an organic solvent; and    -   (4) adjusting the aqueous layer to a pH less than about 6;

In another embodiment, the invention relates to a large-scale processfor forming a compound of formula (XIV)

or a boronic acid anhydride thereof, comprising the steps:

-   -   (aa) coupling a compound of formula (XVIII):

or an acid addition salt thereof, with a compound of formula (XIX):

wherein:

-   -   P¹ is a cleavable amino group protecting moiety; and    -   X is OH or a leaving group;        to form a compound of formula (XX):

wherein P¹ is as defined above, said coupling step (aa) comprising thesteps:

-   -   (i) coupling the compound of formula (XVIII) with a compound of        formula (XIX) wherein X is OH in the presence of        2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium        tetrafluoroborate (TBTU) and a tertiary amine in        dichloromethane;    -   (ii) performing a solvent exchange to replace dichloromethane        with ethyl acetate; and    -   (iii) performing an aqueous wash of the ethyl acetate solution;    -   (bb) removing the protecting group P¹ to form a compound of        formula (XXI):

or an acid addition salt thereof, said protecting group removing step(bb) comprising the steps:

-   -   (i) treating the compound of formula (XX) with HCl in ethyl        acetate;    -   (ii) adding heptane to the reaction mixture; and    -   (iii) isolating by crystallization the compound of formula (XXI)        as its HCl addition salt;    -   (cc) coupling the compound of formula (XXI) with a reagent of        formula (XXII)

wherein X is a OH or a leaving group, to form a compound of formula(XXIII):

said coupling step (cc) comprising the steps:

-   -   (i) coupling the compound of formula (XXI) with        2-pyrazinecarboxylic acid in the presence of TBTU and a tertiary        amine in dichloromethane;    -   (ii) performing a solvent exchange to replace dichloromethane        with ethyl acetate; and    -   (iii) performing an aqueous wash of the ethyl acetate solution;        and    -   (dd) deprotecting the boronic acid moiety to form the compound        of formula (XIV) or a boronic acid anhydride thereof, said        deprotecting step (dd) comprising the steps:        -   (i) providing a biphasic mixture comprising the compound of            formula (XXIII), an organic boronic acid acceptor, a lower            alkanol, a C₅₋₈ hydrocarbon solvent, and aqueous mineral            acid;        -   (ii) stirring the biphasic mixture to afford the compound of            formula (XIV);        -   (iii) separating the solvent layers; and        -   (iv) extracting the compound of formula (XIV), or a boronic            acid anhydride thereof, into an organic solvent.

Preferably, step (dd)(iii) comprises the steps:

-   -   (1) separating the solvent layers;    -   (2) adjusting the aqueous layer to basic pH;    -   (3) washing the aqueous layer with an organic solvent; and    -   (4) adjusting the aqueous layer to a pH less than about 6;

The efficiency of the processes described above is further enhanced bytelescoping steps, for example, by carrying a reaction mixture orworked-up product solution from one reaction directly into the followingreaction, without isolation of the intermediate product. For example, insome embodiments, step (e)(iii) or (aa)(iii) affords an ethyl acetatesolution comprising a compound of formula (XX), and the ethyl acetatesolution is directly subjected in step (f) or (bb) to conditionseffective to remove the protecting group P¹. In some such embodiments,the protecting group P¹ is an acid-labile protecting group, for example,tert-butoxycarbonyl (Boc), and the ethyl acetate solution from step(e)(iii) or (aa)(iii) is treated with acid. In certain preferredembodiments, the ethyl acetate solution from step (e)(iii) or (aa)(iii)is dried azeotropically and then treated with gaseous HCl.

When the deprotecting step (f) or (bb) is performed under anhydrousconditions, as described above, the product of formula (XXI) can beisolated by crystallization from the reaction mixture as its HCladdition salt. Crystallization of the product salt is promoted byaddition of a hydrocarbon solvent such as n-heptane. In someembodiments, the reaction mixture is partially concentrated prior toaddition of the hydrocarbon solvent. The present inventors havediscovered that crystallization of the compound of formula (XXI) in thismanner efficiently removes any tripeptide impurity that may have formedduring the coupling step (e) or (aa). Such impurities are difficult toremove at later stages in the synthesis.

Further telescoping of the process is possible by carrying the productmixture from the coupling step (g) or (cc) directly into the boronicacid moiety deprotecting step (h) or (dd). Preferably, the organicsolvent from the coupling reaction is first replaced with ethyl acetatein order to facilitate aqueous washes. A second solvent exchange into ahydrocarbon solvent then permits the product solution from step (g) or(cc) to be used directly in the biphasic boronic acid deprotecting step(h) or (dd), without isolation of the compound of formula (XXIII).

Alternatively, a more convergent approach may be adopted for thesynthesis of the compound of formula (XIV). Thus, in yet anotherembodiment, the invention provides a large-scale process for forming acompound of formula (XIV)

or a boronic acid anhydride thereof. The process comprises the steps:

-   -   (a) providing a boron “ate” complex of formula (XV):

wherein:

-   -   R³ is a nucleofugic group;    -   Y is a nucleofugic group; and    -   M⁺ is an alkali metal;    -   (b) contacting the boron “ate” complex of formula (XV) with a        Lewis acid under conditions that afford a boronic ester compound        of formula (XVI):

said contacting step being conducted in a reaction mixture comprising:

-   -   (i) a coordinating ether solvent that has low miscibility with        water; or    -   (ii) an ether solvent that has low miscibility with water and a        coordinating co-solvent;    -   (c) treating the boronic ester compound of formula (XVI) with a        reagent of formula M¹-N(Si(R⁶)₃)₂, where M¹ is an alkali metal        and each R⁶ independently is selected from the group consisting        of alkyl, aralkyl, and aryl, where the aryl or aryl portion of        the aralkyl is optionally substituted, to form a compound of        formula (XVII):

-   -   (d) removing the (R⁶)₃Si groups to form a compound of formula        (XVIII):

or an acid addition salt thereof;

-   -   (e′) coupling the compound of formula (XVIII) with a compound of        formula (XIXa):

wherein X is OH or a leaving group, to form a compound of formula(XXIII):

and

-   -   (f′) deprotecting the boronic acid moiety to form the compound        of formula (XIV) or a boronic acid anhydride thereof.

In some embodiments, the process is characterized by at least one of thefollowing features (1)-(3). In certain preferred embodiments, theprocess is characterized by all three features (1)-(3) below.

-   -   (1) In the boron “ate” complex of formula (XV), R³ and Y both        are chloro.    -   (2) The coupling step (e′) comprises the steps:        -   (i) coupling the compound of formula (XVIII) with a compound            of formula (XIXa) wherein X is OH in the presence of            2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium            tetrafluoroborate (TBTU) and a tertiary amine in            dichloromethane;        -   (ii) performing a solvent exchange to replace            dichloromethane with ethyl acetate; and        -   (iii) performing an aqueous wash of the ethyl acetate            solution.    -   (3) The boronic acid deprotecting step (f′) comprises the steps:        -   (i) providing a biphasic mixture comprising the compound of            formula (XXIII), an organic boronic acid acceptor, a lower            alkanol, a C₅₋₈ hydrocarbon solvent, and aqueous mineral            acid;        -   (ii) stirring the biphasic mixture to afford the compound of            formula (XIV);        -   (iii) separating the solvent layers; and        -   (iv) extracting the compound of formula (XIV), or a boronic            acid anhydride thereof, into an organic solvent.

Preferably, step (f′)(iii) comprises the steps:

-   -   (1) separating the solvent layers;    -   (2) adjusting the aqueous layer to basic pH;    -   (3) washing the aqueous layer with an organic solvent; and    -   (4) adjusting the aqueous layer to a pH less than about 6;

In step (h)(iv), (dd)(iv), or (f′)(iv) of the processes described above,the compound of formula (XIV), or a boronic acid anhydride thereof,preferably is extracted into ethyl acetate and crystallized by additionof hexane or heptane. In some embodiments, the process further comprisesisolation of a boronic acid anhydride of the compound of formula (XIV),preferably a trimeric boronic acid anhydride of formula (XXIV):

The processes of the invention permit the large-scale manufacture ofbortezomib of very high chemical and stereochemical purity. Prior artprocesses were limited in scale and afforded product of lower overallpurity. In yet another aspect, therefore, the invention provides acomposition comprising at least one kilogram of a compound of formula(XXIV):

The compound of formula (XXIV) preferably is prepared according to theprocess described above, and preferably constitutes at least 99% w/w ofthe composition according to this aspect of the invention.

EXAMPLES Abbreviations

BOC tert-butoxycarbonylD.I. de-ionized

DMF N,N-dimethylformamide

GC gas chromatographyGC-MS gas chromatography-mass spectrometryh hoursHDPE high density polyethyleneHPLC high performance liquid chromatographyLDA lithium diisopropylamideLOD loss on dryingmin minutesMTBE t-butyl methyl etherRP-HPLC reverse phase high performance liquid chromatographyRPM revolutions per minuteTBTU O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium tetrafluoroborateTHF tetrahydrofuran

Example 1: (1R)—(S)-Pinanediol 1-ammoniumtrifluoroacetate-3-methylbutane-1-boronate Manufacturing Process(1S)—(S)-Pinanediol 1-chloro-3-methylbutane-1-boronate

-   1. (S)-Pinanediol-2-methylpropane-1-boronate (12.0 kg, 50.8 moles)    was charged to a reaction vessel maintained under a nitrogen    atmosphere.-   2. tert-Butyl methyl ether (53 kg) and dichloromethane (22.5 kg)    were charged and the resultant mixture was cooled to −57° C. with    stirring.-   3. Diisopropylamine, (6.7 kg) was charged to another reaction vessel    maintained under a nitrogen atmosphere.-   4. tert-Butyl methyl ether (27 kg) was charged to the    diisopropylamine and the resultant mixture was cooled to −10° C.    with stirring.-   5. n-Hexyllithium in hexane (33.2 weight % solution) (17.6 kg) was    added to the diisopropylamine mixture over a period of 57 minutes,    while the reaction temperature was maintained at −10° C. to −7° C.-   6. This mixture (LDA-mixture) was stirred for 33 minutes at −9° C.    to −7° C. before it was used.-   7. Zinc chloride, (12.1 kg) was charged to a third reaction vessel    maintained under a nitrogen atmosphere.-   8. tert-Butyl methyl ether (16 kg) was charged to the zinc chloride    and the resultant mixture was warmed to 30° C. with stirring.-   9. Tetrahydrofuran (53 kg) was added to the zinc chloride suspension    over a period of 18 minutes, while the reaction temperature was    maintained at 35° C. to 40° C.-   10. This mixture (ZnCl₂-mixture) was stirred for 4 hours and 28    minutes at 38° C. to 39° C. until it was used.-   11. The LDA-mixture (from #3-6) was added over a period of 60    minutes to the reaction vessel containing    (S)-pinanediol-2-methylpropane-1-boronate, while the reaction    temperature was maintained at −60° C. to −55° C.-   12. A tert-butyl methyl ether rinse (10 kg) was used to complete the    addition.-   13. The reaction mixture was stirred for an additional 20 minutes at    −59° C. to −55° C.-   14. The reaction mixture was warmed to −50° C. over a period of 11    minutes.-   15. The ZnCl₂-mixture (from #7-10) was added over a period of 48    minutes to the reaction vessel containing    (S)-pinanediol-2-methylpropane-1-boronate and the LDA-mixture, while    the reaction temperature was maintained at −50° C. to −45° C.-   16. A tert-butyl methyl ether rinse (10 kg) was used to complete the    addition.-   17. The reaction mixture was stirred for an additional 30 minutes at    −45° C. to −40° C. and then warmed to 10° C. over a period of 81    minutes.-   18. A 10% sulfuric acid solution (72 kg) was added over a period of    40 minutes to the reaction vessel, while the reaction temperature    was maintained at 10° C. to 21° C.-   19. The reaction mixture was stirred for 16 minutes at ambient    temperature, before the aqueous phase was separated.-   20. The organic phase was washed successively with deionized (D.I.)    water (32 kg), and 10% sodium chloride solution (26.7 kg), each wash    involved vigorous stirring for 15 to 17 minutes at ambient    temperature.-   21. The reaction mixture was concentrated under reduced pressure    (p_(min)=81 mbar), maintaining an external (jacket/bath) temperature    of 50° C. to 55° C., providing a residue which was dissolved in    methylcyclohexane (56 kg).-   22. The reaction mixture was refluxed (in a Dean-Stark type    condenser for water separation) under reduced pressure (p_(min)=67    mbar), maintaining an external (jacket/bath) temperature of 50° C.    to 55° C. for 2 hours and 7 minutes, until no more water was    separated.-   23. About 35 L of the solvents were distilled off under reduced    pressure (p_(min)=81 mbar), maintaining an external (jacket/bath)    temperature of 50° C. to 55° C.-   24. The resultant dry methylcyclohexane mixture containing    (1S)—(S)-pinanediol 1-chloro-3-methylbutane-1-boronate was cooled to    14° C.

(1R)—(S)-Pinanediol 1-bis(trimethylsilyl)amino-3-methylbutane-1-boronate

-   1. Lithium bis(trimethylsilyl)amide in tetrahydrofuran (19.4 weight    % solution), (41.8 kg) was charged to a reaction vessel maintained    under a nitrogen atmosphere and cooled to −19° C. with stirring.-   2. The methylcyclohexane mixture containing (1S)—(S)-pinanediol    1-chloro-3-methylbutane-1-boronate was added over a period of 55    minutes, while the reaction temperature was maintained at −19° C. to    −13° C.-   3. A methylcyclohexane rinse (5 kg) was used to complete the    addition.-   4. The reaction mixture was stirred for an additional 65 minutes at    −13° C. to −12° C. and then warmed to 25° C. over a period of 25    minutes.-   5. A suspension of Celite (2.5 kg) in methylcyclohexane (22 kg) was    added to the reaction mixture.-   6. The reaction mixture was concentrated under reduced pressure    (p_(min)=25 mbar), maintaining an external (jacket/bath) temperature    of 45° C. to 50° C., providing a residue which was dissolved in    methylcyclohexane (36 kg).-   7. A sample was then removed for in-process testing for    tetrahydrofuran content by GC.-   8. The tetrahydrofuran assay was 0.58%.-   9. The solids were removed by filtration, the filtrate was filtered    through a plug of Silica Gel (2.0 kg).-   10. Both filter units were washed with isopropyl ether (30 kg).-   11. The resultant methylcyclohexane/isopropyl ether mixture    containing (1R)—(S)-pinanediol    1-bis(trimethylsilyl)amino-3-methylbutane-1-boronate was stored in a    container at ambient temperature until it was used in the next step.

(1R)—(S)-Pinanediol 1-ammoniumtrifluoroacetate-3-methylbutane-1-boronate

-   1. Trifluoroacetic acid, (12 kg) was charged to another reaction    vessel maintained under a nitrogen atmosphere.-   2. Isopropyl ether (78 kg) was charged to the trifluoroacetic acid    and the resultant mixture was cooled to −10° C. with stirring.-   3. The methylcyclohexane/isopropyl ether mixture containing    (1R)—(S)-pinanediol    1-bis(trimethylsilyl)amino-3-methylbutane-1-boronate was added over    a period of 53 minutes causing product precipitation, while the    reaction temperature was maintained at −10° C. to −5° C.-   4. An isopropyl ether rinse (5 kg) was used to complete the    addition.-   5. The reaction mixture was stirred for an additional 8 hours and 20    minutes at −9° C. to −7° C.-   6. The solid was collected by filtration, washed with isopropyl    ether (70 kg) in two portions, and dried under reduced pressure    (pmin=56 mbar) at 41° C. to 42° C. for 2 hours and 15 minutes.-   7. The solid was stirred with D.I. water (60 kg) for 24 minutes at    ambient temperature, before the D.I. water was removed by    filtration.-   8. The solid was washed with D.I. water (12 kg).-   9. The solid was then dried under vacuum (pmin=4 mbar) at 40° C. to    44° C. for 9 hours and 22 minutes, after that time the loss on    drying was 0.51%, which meets the ≤1% requirement.-   10. The intermediate, (1R)—(S)-pinanediol 1-ammonium    trifluoroacetate-3-methylbutane-1-boronate, crude, was then packaged    into single polyethylene bags in polypropylene drums and labeled.    The yield was 72%.

Recrystallization of (1R)—(S)-pinanediol 1-ammoniumtrifluoroacetate-3-methylbutane-1-boronate, Crude

-   1. (1R)—(S)-Pinanediol 1-ammonium    trifluoroacetate-3-methylbutane-1-boronate, crude, (13 kg) was    charged to a reaction vessel maintained under a nitrogen atmosphere.-   2. Trifluoroacetic acid (31 kg) was charged to the reaction vessel    and the resultant mixture was cooled to 4° C. with stirring.-   3. Once all of the solid was dissolved leaving a slightly turbid    mixture, isopropyl ether (29 kg) was added over a period of 57    minutes, while the reaction temperature was maintained at 2° C. to    3° C.-   4. After complete addition the mixture was filtered through a filter    into a receiving vessel maintained under a nitrogen atmosphere.-   5. Reactor and filter were rinsed with a mixture of trifluoroacetic    acid (3.8 kg) and isopropyl ether (5 kg). The rinse was added to the    filtrate.-   6. Isopropyl ether (126 kg) was added over a period of 15 minutes    causing product precipitation, while the reaction temperature was    maintained at 16° C. to 18° C.-   7. The mixture was stirred at 16° C. to 18° C. for 15 min, then    cooled to −5° C. over a period of 67 minutes, and stirred at −3° C.    to −5° C. under a nitrogen atmosphere for 89 minutes.-   8. The solid was then isolated by filtration, washed with isopropyl    ether (48 kg) in two portions, and dried under vacuum (pmin=2 mbar)    at 34° C. to 40° C. for 2 hours and 55 minutes after that time the    loss on drying was 0.32%, which meets the ≤0.5% requirement.-   9. The product, (1R)—(S)-pinanediol 1-ammonium    trifluoroacetate-3-methylbutane-1-boronate, was then packaged into    double polyethylene bags in fiber drums and labeled. The yield was    86%.

Example 2: N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine boronicAnhydride Manufacturing Process (1S,2S,3R,5S)-PinanediolN—BOC-L-phenylalanine-L-leucine Boronate

-   1. In a fume hood, a three-necked glass reaction flask equipped with    a Claisen head temperature recorder and a mechanical stirrer was    flushed with nitrogen.-   2. (1R)—(S)-Pinanediol 1-ammonium    trifluoroacetate-3-methylbutane-1-boronate (2.0 kg), was charged to    the flask.-   3. BOC-L-phenylalanine (1.398 kg) was charged to the flask.-   4. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium    tetrafluoroborate, TBTU (1.864 kg) was charged to the flask.-   5. Dichloromethane (15.8 L) was charged to the flask.-   6. The stirring motor was adjusted to provide stirring at 260 RPM.-   7. Using an ice/water cooling bath, the reaction mixture was cooled    to 1.0° C., maintaining a nitrogen atmosphere.-   8. N,N-Diisopropylethylamine (2.778 L) was charged to a glass flask    and transferred to the reaction mixture over a period of 117 minutes    using a peristaltic pump maintaining a reaction temperature range of    0.7° C.-2.1° C. The overall addition rate was 23.7 mL/min.-   9. A dichloromethane (0.2 L) rinse of the flask into the reaction    mixture was used to complete the addition.-   10. The reaction mixture was stirred for an additional 35 minutes.    The temperature at the start of the stir time was 1.8° C., and    2.5° C. at the end.-   11. A sample was then removed for in-process testing by reverse    phase high performance liquid chromatography (RP-HPLC). The percent    conversion was determined to be 99.3%.-   12. The reaction mixture was transferred in approximately two equal    halves to two rotary evaporator flasks. The reaction mixture was    concentrated under reduced pressure using a rotary evaporator,    maintaining an external bath temperature of 29-30° C.-   13. Ethyl acetate (4.0 L) was divided into two approximately equal    portions and charged to the two rotary evaporator flasks.-   14. The mixtures in each flask were again concentrated under reduced    pressure using a rotary evaporator, maintaining an external bath    temperature of 29-30° C.-   15. The residues in each rotary evaporator flask were then    transferred back to the reaction flask using ethyl acetate (13.34    L).-   16. In a glass flask equipped with a stirrer, a 1% aqueous    phosphoric acid solution was prepared by mixing D.I. water (13.18 L)    and phosphoric acid (0.160 kg).-   17. In a glass flask equipped with a stirrer, a 2% aqueous potassium    carbonate solution (12.0 L) was prepared by mixing D.I. water    (11.76 L) and potassium carbonate (0.24 kg).-   18. In a glass flask equipped with a stirrer, a 10% aqueous sodium    chloride solution (13.34 L) was prepared by mixing D.I. water    (13.34 L) and sodium chloride (1.334 kg).-   19. D.I. water (13.34 L) was charged to the reaction flask    containing the ethyl acetate solution and the mixture stirred at 380    RPM for 7 minutes. The layers were allowed to separate and the    aqueous phase (bottom layer) was transferred under vacuum to a    suitable flask and discarded.-   20. Again, D.I. water (13.34 L) was charged to the reaction flask    containing the ethyl acetate solution and the mixture stirred at 385    RPM for 7 minutes. The layers were allowed to separate and the    aqueous phase (bottom layer) was transferred under vacuum to a    suitable flask and discarded.-   21. The 1% phosphoric acid solution prepared in Step 16 was charged    to the reaction flask containing the ethyl acetate solution and the    mixture stirred at 365 RPM for 7 minutes. The layers were allowed to    separate and the acidic aqueous phase (bottom layer) was transferred    to a suitable flask and discarded.-   22. The 2% potassium carbonate solution prepared in Step 17 was    charged to the reaction flask containing the ethyl acetate solution    and the mixture stirred at 367 RPM for 7 minutes. The layers were    allowed to separate and the basic aqueous phase (bottom layer) was    transferred to a suitable flask and discarded.-   23. The 10% sodium chloride solution prepared in Step 18 was charged    to the reaction flask containing the ethyl acetate solution and the    mixture stirred at 373 RPM for 6 minutes. The layers were allowed to    separate and the aqueous phase (bottom layer) was transferred to a    suitable flask and discarded.-   24. The ethyl acetate solution was transferred to a rotary    evaporator flask and concentrated under reduced pressure using a    rotary evaporator, maintaining a bath temperature of 29-30° C., to    provide a residue.-   25. The residue was then redissolved in ethyl acetate (4.68 L).-   26. The solution was concentrated under vacuum using a rotary    evaporator, maintaining a bath temperature of 29-30° C., to provide    a residue once more.-   27. Again, the residue was then redissolved in ethyl acetate    (4.68 L) and two samples taken for determination of water content by    Karl Fisher titration. The water content of two samples was    determined as 0.216% and 0.207%.-   28. Using a further quantity of ethyl acetate (12.66 L), the mixture    was transferred from the rotary evaporator flask to a dry reaction    flask equipped with a temperature recorder, a mechanical stirrer,    and a fritted gas dispersion tube, and purged with nitrogen.

(1S,2S,3R,5S)-Pinanediol L-phenylalanine-L-leucine Boronate, HCl Salt

-   1. The ethyl acetate solution containing (1S,2S,3R,5S)-pinanediol    N—BOC-L-phenylalanine-L-leucine boronate was cooled using an    ice/water cooling bath to −0.9° C.-   2. Hydrogen chloride (1.115 kg) gas was bubbled into the reaction    mixture over a period of 1.48 hours. The temperature at the start of    the addition was −0.9° C., and 6.8° C. at the end.-   3. The reaction was then allowed to warm to 14.4° C. over 50    minutes, while maintaining a nitrogen atmosphere.-   4. A sample was removed for in-process testing by RP-HPLC. The    percent conversion was 68.9% (area %).-   5. The reaction was stirred for 35 minutes. The temperature at the    start was 14° C., and 14.8° C. at the end.-   6. A sample was removed for in-process testing by RP-HPLC. The    percent conversion was 94.7% (area %).-   7. The reaction was stirred for approximately a further 50 minutes,    maintaining a temperature of 10° C.±5° C.-   8. A sample was removed for in-process testing by RP-HPLC. The    percent conversion was 97.3%.-   9. The reaction was stirred for approximately a further 50 minutes,    maintaining a temperature of 10° C.±5° C. The final temperature was    14.6° C.-   10. A sample was removed for in-process testing by RP-HPLC. The    total reaction time after addition of hydrogen chloride gas was    four (4) hours.-   11. The percent conversion was 99%.-   12. A slurry was observed.-   13. n-Heptane (8.8 L) was charged to the reaction mixture.-   14. The slurry was stirred for 2 hours. The temperature at the start    of the stir time was 12.7° C., and 15.3° C. at the end.-   15. The solid was isolated by filtration on a Buchner funnel lined    with a polypropylene felt filter pad.-   16. The solid was washed with n-heptane (4.68 L).-   17. In a hood, the solid was transferred to three drying trays at    not more than 1″ deep and air-dried for 1 hour.-   18. The solid was then dried at ≤35° C. under a vacuum of 27″ of Hg    for 16 hours 28 minutes in a vacuum oven equipped with a vacuum    gauge and a temperature recorder.-   19. The solid was sampled from each drying tray to determine the %    Loss on Drying. The LOD was determined to be 0%, 0.02%, and 0.02% on    the three samples taken.-   20. (1S,2S,3R,5S)-Pinanediol L-phenylalanine-L-leucine boronate, HCl    salt was then packaged into double poly bags in fiber drums and    labeled, and sampled.-   21. The isolated yield was 1.87 kg, 79.1%. The intermediate was    stored at 2-8° C. until used in further manufacturing.

(1S,2S,3R,5S)-PinanediolN-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine Boronate

-   1. In a fume hood a three-necked glass reaction flask equipped with    a Claisen head, temperature recorder and a mechanical stirrer was    flushed with nitrogen.-   2. (1S,2S,3R,5S)-Pinanediol L-phenylalanine-L-leucine boronate, HCl    salt (1.85 kg) was charged to the flask.-   3. 2-Pyrazinecarboxylic acid (0.564 kg) was charged to the flask.-   4. 2-(H-Benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium    tetrafluoroborate, TBTU (1.460 kg) was charged to the flask.-   5. Dichloromethane (18.13 L) was charged to the flask.-   6. The stirring motor was adjusted to provide stirring at 272 RPM.-   7. Using a cooling bath, the reaction mixture was cooled to −1.2° C.-   8. N,N-Diisopropylethylamine (1.865 kg) was charged to a glass flask    and transferred to the reaction over a period of 50 minutes using a    peristaltic pump maintaining a reaction temperature range of    −1.2° C. to 2.8° C.-   9. A dichloromethane rinse (0.37 L) of the flask into the reaction    mixture was used to complete the addition.-   10. The reaction mixture was allowed to warm and stirred for an    additional 81 minutes.-   11. The temperature at the start of the stir time was 15° C., and    24.9° C. at the end.-   12. A sample was then removed for in-process testing by RP-HPLC. The    percent conversion was determined to be 99.9%.-   13. The reaction mixture was transferred in approximately two equal    halves to two rotary evaporator flasks. The reaction mixture was    concentrated under reduced pressure using two rotary evaporators,    maintaining an external bath temperature of 33-34° C.-   14. Ethyl acetate (12.95 L) was divided into two approximately equal    portions and charged to the two rotary evaporator flasks.-   15. The mixtures in each flask were then concentrated under reduced    pressure using a rotary evaporator, maintaining an external bath    temperature of 33-34° C.-   16. The residues in each rotary evaporator flask were then    transferred back to the reaction flask using ethyl acetate (12.95    L).-   17. In a glass flask equipped with a stirrer, a 1% aqueous    phosphoric acid solution (12.34 L) was prepared by mixing D.I. water    (12.19 L) and phosphoric acid (0.148 kg).-   18. In a glass flask equipped with a stirrer, a 2% aqueous potassium    carbonate solution (12.34 L) was prepared by mixing D.I. water    (12.09 L) and potassium carbonate (0.247 kg).-   19. In a glass flask equipped with a stirrer, a 10% aqueous sodium    chloride solution (12.34 L) was prepared by mixing D.I. water    (12.34 L) and sodium chloride (1.234 kg).-   20. D.I. water (12.34 L) was charged to the reaction flask    containing the ethyl acetate solution and the mixture stirred at 382    RPM for 7 minutes. The layers were allowed to separate and the    aqueous phase (bottom layer) was transferred to a suitable flask and    discarded.-   21. Again, D.I. water (12.34 L) was charged to the reaction flask    containing the ethyl acetate solution and the mixture stirred at 398    RPM for 7 minutes. The layers were allowed to separate and the    aqueous phase (bottom layer) was transferred to a suitable flask and    discarded.-   22. The 1% phosphoric acid solution prepared in Step 17 was charged    to the reaction flask containing the ethyl acetate solution and the    mixture stirred at 364 RPM for 8 minutes. The layers were allowed to    separate and the acidic aqueous phase (bottom layer) was transferred    to a suitable flask and discarded.-   23. The 2% potassium carbonate solution prepared in Step 18 was    charged to the reaction flask containing the ethyl acetate solution    and the mixture stirred at 367 RPM for 8 minutes. The layers were    allowed to separate and the basic aqueous phase (bottom layer) was    transferred to a suitable flask and discarded.-   24. The 10% sodium chloride solution prepared in Step 19 was charged    to the reaction flask containing the ethyl acetate solution and the    mixture stirred at 374 RPM for 8 minutes. The layers were allowed to    separate and the aqueous phase (bottom layer) was transferred to a    suitable flask and discarded.-   25. The ethyl acetate solution was transferred under vacuum in    approximately two equal halves to two rotary evaporator flasks and    concentrated under reduced pressure using a rotary evaporator,    maintaining an external bath temperature of 34° C.-   26. n-Heptane (14.8 L) was divided into two approximately equal    portions and charged to the two rotary evaporator flasks. The    mixtures in each flask were then concentrated under reduced pressure    using a rotary evaporator, maintaining an external bath temperature    of 34° C.

N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine Boronic Anhydride,Crude

-   1. In a glass flask equipped with a stirrer, a 1N solution of    hydrochloric acid (22.2 L) was prepared by mixing D.I. water    (20.36 L) and hydrochloric acid (1.84 kg).-   2. In a glass flask equipped with a stirrer, a 2N sodium hydroxide    solution (12.03 L) was prepared by mixing D.I. water (12.03 L) and    sodium hydroxide (0.962 kg).-   3. The residues containing (1S,2S,3R,5S)-pinanediol    N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronate in each    rotary evaporator flask were then transferred to a three-necked    glass reaction flask equipped with a temperature recorder and a    mechanical stirrer, using n-heptane (14.8 L) and methanol (14.8 L).-   4. The stirring motor was adjusted to provide stirring at 284 RPM.-   5. 2-Methylpropaneboronic acid (0.672 kg) was charged to the flask.-   6. 1N hydrochloric acid prepared in Step 1 (11.2 L) was charged to    the flask.-   7. The stirring motor was adjusted to provide stirring at 326 RPM.-   8. The reaction mixture was stirred for 16.38 hours The start batch    temperature was 28.6° C., and the end batch temperature was 21.6° C.-   9. A sample was then removed for in-process testing by RP-HPLC.-   10. The percent conversion was determined to be 100%.-   11. Stirring was stopped and the biphasic mixture allowed to    separate.-   12. The n-heptane layer (upper layer) was transferred to a suitable    flask and discarded.-   13. n-Heptane (5.37 L) was charged to the reaction flask and the    mixture stirred at 381 RPM for 6 minutes. The layers were allowed to    separate and the n-heptane phase (upper layer) was transferred to a    suitable flask and discarded.-   14. Again, n-heptane (5.37 L) was charged to the reaction flask and    the mixture stirred at 340 RPM for 6 minutes. The layers were    allowed to separate and the n-heptane phase (upper layer) was    transferred to a suitable flask and discarded.-   15. The aqueous methanol solution was transferred in approximately    two equal halves to two rotary evaporator flasks and concentrated    under reduced pressure using a rotary evaporator, maintaining an    external bath temperature of 33-34° C. 15 L of methanol were    collected.-   16. Dichloromethane (5.37 L) was used to transfer the residue from    the rotary evaporator flasks back into the reaction flask.-   17. 2N sodium hydroxide (11.2 L) prepared in Step 2 was charged to    the flask.-   18. The dichloromethane layer (lower layer) was transferred to a    suitable flask and discarded.-   19. Dichloromethane (5.37 L) was charged to the flask and the    mixture stirred at 374 RPM for 6 minutes. The phases were allowed to    separate and the dichloromethane layer (lower layer) was transferred    to a suitable flask and discarded.-   20. Again, dichloromethane, (5.37 L) was charged to the flask and    the mixture stirred at 368 RPM for 8 minutes. The phases were    allowed to separate and the dichloromethane layer (lower layer) was    transferred to a suitable flask and discarded.-   21. Dichloromethane (5.37 L) was charged to the flask.-   22. 1N hydrochloric acid (10.7 L) was charged to the flask with    stirring. The pH of the aqueous phase was determined to be 6.-   23. Stirring was discontinued and the phases allowed to separate.-   24. The dichloromethane phase (lower layer) was transferred under    vacuum to a glass receiving flask.-   25. Dichloromethane (5.37 L) was charged to the flask and the    mixture stirred at 330 RPM for 6 minutes. The phases were allowed to    separate and the dichloromethane layer (lower layer) was transferred    to the glass receiving flask.-   26. Again, dichloromethane, (5.37 L) was charged to the flask and    the mixture stirred at 335 RPM for 6 minutes. The phases were    allowed to separate and the dichloromethane layer (lower layer) was    transferred to the glass receiving flask.-   27. The dichloromethane extracts were combined and transferred in    approximately two equal halves to two rotary evaporator flasks and    concentrated under reduced pressure using a rotary evaporator,    maintaining an external bath temperature of 33-34° C.-   28. Ethyl acetate (12.95 L) was divided into two approximately equal    portions and charged to the two rotary evaporator flasks. The    mixtures in each flask were then concentrated under reduced pressure    using a rotary evaporator, maintaining an external bath temperature    of 45-46° C.-   29. Again, ethyl acetate (12.95 L) was divided into two    approximately equal portions and charged to the two rotary    evaporator flasks. The mixtures in each flask were then concentrated    under reduced pressure using a rotary evaporator, maintaining an    external bath temperature of 45-46° C., until approximately 10% of    the original volume remained.-   30. n-Heptane (10.2 L) was divided into two approximately equal    portions and charged to the two rotary evaporator flasks, and the    slurry stirred under a nitrogen atmosphere for 2.67 hours at 22-23°    C.-   31. The solid was isolated by filtration on a Buchner funnel, lined    with a polypropylene felt filter pad.-   32. The solid was washed with n-heptane (2.96 L).-   33. In a hood, the solid was transferred to four drying trays and    air-dried for 1.25 hours.-   34. The solid was then dried at 36-50° C. under a vacuum of 27″ of    Hg for 18 hours 27 minutes in a vacuum oven equipped with a vacuum    gauge and a temperature recorder.-   35. The solid was sampled from each tray to determine the % Loss on    Drying (LOD). The LOD was determined to be 0.38%, 0.62%, 0.71%, and    0.63% on the four samples taken.-   36. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic    anhydride, crude was packaged into two 5 L, HDPE, tamper-proof    wide-mouth bottles and labeled.-   37. The isolated yield was 1.314 kg, 83%.

Recrystallization of N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucineBoronic Anhydride, Crude

-   1. In a hood a glass reaction flask equipped with a mechanical    stirrer, a reflux condenser and a temperature recorder was flushed    with nitrogen.-   2. Ethyl acetate (21 L) was charged to the flask.-   3. The ethyl acetate was heated to 66.8° C. under a nitrogen    atmosphere, using a hot water/steam bath.-   4. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic    anhydride, crude (1.311 kg) was slowly charged to the reaction    flask. Charging occurred over a period of 3 minutes.-   5. The mixture was stirred for 1 minute until all the solid had    dissolved. The temperature of the solution was 64° C.-   6. The heat source was removed and the mixture was slowly cooled to    60° C. using a cold bath.-   7. The hot ethyl acetate solution was transferred into a receiving    flask via poly tubing and a polypropylene in-line filter capsule    using a peristaltic pump.-   8. The mixture was allowed to cool to 27.2° C., and allowed to stand    under a nitrogen atmosphere without stirring, for 17.75 hours. The    final temperature was recorded as 20.5° C.-   9. The mixture was cooled using an ice/water bath with stirring for    2.33 hours. The temperature at the start of the stir time was 3.8°    C., and −2.8° C. at the end.-   10. The solid was isolated by filtration on a Buchner funnel lined    with a polypropylene felt filter pad. The filtrate was collected in    a collection flask.-   11. The solid was washed with ethyl acetate (2.62 L), cooled to 4.7°    C.-   12. In a hood, the solid was transferred to two drying trays.-   13. The solid was then dried at 51-65° C. under a vacuum of 27″ of    Hg for 19 hours 10 minutes in a vacuum oven equipped with a vacuum    gauge and a temperature recorder.-   14. The solid was sampled to determine the % Loss on Drying (LOD).    The LOD was determined to be 0.65% and 0.62% on the two samples    taken.-   15. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic    anhydride was packaged into four 1 L, Type 3, Amber Wide-Mouth    Bottles with Teflon-Lined Caps and labeled.-   16. The isolated yield was 1.132 kg, 86.3%.-   17. N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic    anhydride was stored at −25 to −15° C.

Example 3: N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine BoronicAnhydride Convergent Synthesis (1S,2S,3R,5S)-PinanediolN-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine Boronate

A solution of (1R)—(S)-Pinanediol 1-ammoniumtrifluoroacetate-3-methylbutane-1-boronate (13.97 g) andN-hydroxysuccinimide (6.23 g) of in 66 mL of DMF was cooled to −5° C.,followed by the addition of dicyclohexylcarbodiimide (10.83 g). Theresulting suspension was stirred for one hour at a temperature of −5 to0° C. To a solution of N-(2-pyrazinecarbonyl)-L-phenylalanine (19.52 g;prepared by coupling the preformed succinimide ester ofpyrazinecarboxylic acid with L-phenylalanine in dioxane-water) in 62 mLof DMF was added N-methylmorpholine (5.7 mL) at a temperature of 0° C.,and the resulting solution was added to the suspension. The suspensionwas adjusted to pH 7 by the addition of another 5.7 mL ofN-methylmorpholine and stirred overnight, raising the temperature slowlyto 21° C. After filtration, the filtercake was washed twice with MTBEand the combined filtrates were diluted with 950 mL of MTBE. The organiclayer was washed with 20% aqueous citric acid (3×150 mL), 20% aqueousNaHCO₃ (3×150 mL), and brine (2×). The organic layer was dried overNa₂SO₄, filtered, and concentrated, yielding 25.5 g (95.5%) of the titlecompound as a foam. As indicated by tlc this material contained someminor impurities, including approximately 2% of cyclohexyl urea.

N-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine Boronic Anhydride

A solution of (1S,2S,3R,5S)-PinanediolN-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronate (25.2 g) in207 mL of MeOH and 190 mL of hexane was cooled to 15° C., and 109.4 mLof 1N HCl were added in portions, keeping the temperature between 15 and25° C. 2-Methylpropaneboronic acid (8.67 g) was then added undervigorous stirring, and the stirring of the biphasic mixture wascontinued over night. After separation of the two phases, the lowerlayer was extracted once with 75 mL of hexane. The lower layer was thenconcentrated in vacuo until it became cloudy, followed by the additionof 109.4 mL of 2N NaOH and 100 mL of Et₂O. The two phases were separatedthe lower layer was extracted with Et₂O (4×100 mL each), and thenbrought to pH 6.0 by the addition of 109 mL of 1N HCl. After extractionwith 100 mL of ethyl acetate, the lower layer was adjusted to pH 6.0with 1N HCl and extracted one more time with 75 mL of ethyl acetate. Thecombined ethyl acetate layers were washed with semi-saturated brine(2×25 mL) and brine (2×25 mL), dried over Na₂SO₄, filtered, andconcentrated to afford 15.3 g (81.8%) of crudeN-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine boronic anhydride as afoam. The crude material was dissolved in 150 mL of ethyl acetate andconcentrated in vacuo to a suspension, followed by the addition of 150mL of MTBE. The suspension was stored between 2 and 8° C. over night,filtered, washed twice with MTBE, and dried under high vacuum, yielding10.69 g (57.2%) of N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucineboronic anhydride as a white solid.

Example 4: Measurement of Diastereomeric Ratio of(1R)-(1S,2S,3R,5S)-Pinanediol-1-ammoniumtrifluoroAcetate-3-methylbutane-1-boronate

The diastereomeric purity of(1R)-(1S,2S,3R,5S)-pinanediol-1-ammoniumtrifluoroacetate-3-methylbutane-1-boronate(compound 1) was determined by non-chiral gas chromatography (GC).

-   Chemicals: Acetonitrile (p.a. Bruker or equivalent)    -   Tetradecane (internal standard) (Fluka puriss. or equivalent)    -   Trifluoroacetic anhydride (TFAA) (p.a. Merck or equivalent)-   Instrument: Trace-GC 2000 system or equivalent    -   Mobile phase: H₂    -   Solvent A (with internal standard) Approximately 300 mg of        tetradecane were weighed with an accuracy of 0.1 mg into a        100-mL volumetric flask. 1.5 mL of TFAA were added and the flask        was brought to volume with acetonitrile.-   Sample Preparation: About 150 mg of the sample were exactly weighed    (within 0.1 mg) into a 10-mL volumetric flask. The flask was brought    to volume with Solvent A. The solution was stored for 15 minutes    before injection.-   GC Parameters:    -   Column: Rtx-200; 105 m×0.25 mm i.d.×0.25 m film    -   Mobile phase: H₂    -   Temp. program: 130° C. (0.5 min); 0.5° C./min to 200° C. (0        min); 30° C./min to 300° C. (2 min)    -   Flow: 0.9 mL/min (const. flow)    -   Injector temperature: 250° C.    -   Detector temperature: 250° C. (FID)    -   Split: 1:50    -   Injection volume: 1 μL

Substances

Stability of the Solution

A stock solution of compound 1 was prepared by weighing 150.13 mg ofcompound 1 into a 10-mL volumetric flask and bringing it to volume withSolvent A. Stability of this solution was tested at ambient temperatureover 48 hours. The stock solution was filled in 6 separate GC vials.Injections onto the GC system were carried out from these vials after 0,12, 24, 48, and 72 hours (double injection out of each vial. The area %of compound 1 and compound 2 were determined. No changes in area % wereobserved, indicating that the solution is stable over 72 hours atambient temperature.

Specificity

Approximately 150 mg of a sample comprising compound 1 and compound 2were dissolved in Solvent A and injected to the GC chromatographicsystem. The peak for compound 1 was well separated from the peak forcompound 2. Peak purity check by GC-MS showed no other componentsco-eluting with compound 1 or compound 2.

Limit of Detection

The limit of detection (LOD) was defined to be that concentration wherethe signal of compound 1 showed a signal to noise ratio of at least 3:1.A previous blank measurement was carried out to show that no other peaksinterfered. The signal to noise ratio was calculated by the equation:

${S/N} = \frac{H({signal})}{H({baseline})}$S/N = signal  to  noise  ratioH(signal) = height  of  signal  for  compound  1  [mm]H(baseline) = height  of  signal  baseline  [mm]

A sample concentration of 0.05% of the standard test sampleconcentration was injected and showed a signal to noise ratio of 4.3.Therefore, the limit of detection is 0.0075 mg/mL.

Limit of Quantitation

The limit of quantitation (LOQ) was defined to be that concentrationwhere the signal of compound 1 showed a signal to noise ratio of atleast 10:1. Signal to noise ratio was calculated as described above. Asample concentration of 0.1% of the standard sample concentration wasinjected and showed a signal to noise ratio of 10.1. Therefore, thelimit of quantitation is 0.015 mg/mL.

Example 5: Purity Assay forN-(2-Pyrazinecarbonyl)-L-phenylalanine-L-leucine Boronic Anhydride

The purity of N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucine boronicanhydride (compound 3) was assayed by reverse phase HPLC.

-   Reagents: Water, HPLC grade    -   Acetonitrile, HPLC grade    -   Formic acid, ACS grade, ≥98% pure    -   3% Hydrogen peroxide, ACS grade or equivalent-   Instrument    -   High performance liquid chromatograph Autosampler capable of        delivering 20-υL injections and chromatograph maintaining a        temperature of 5° C.        -   Pump capable of gradient delivery at 1.0 mL/min        -   UV detector capable of monitoring effluent at 270 nm    -   Column Symmetry C18 chromatographic column, 250 mm×4.6 mm ID,        5-μm, Waters, cat # WAT054275.-   Sample Preparation: Approximately 50 mg of compound 3 were    accurately weighed into a 50-mL volumetric flask. Mobile Phase B (5    mL) was added and the mixture was sonicated to dissolve compound 3    (approximately 30-60 seconds). The solution was allowed to reach    room temperature, diluted to volume with Mobile Phase A, and mixed    well. Each sample was prepared in duplicate and was stable for 7    days when stored at 2-8° C. protected from light.-   HPLC Parameters:    -   Mobile phase A: acetonitrile/water/formic acid, 30:70:0.1        (v/v/v), degassed    -   Mobile phase B: acetonitrile/water/formic acid, 80:20:0.1        (v/v/v), degassed    -   Flow rate: 1.0 mL/min    -   Detector: UV at 270 nm    -   Injection Volume: 20 μL    -   Column Temp: ambient    -   Sample Tray Temp: 5° C.

Gradient Program: Time % A % B 0 100 0 15 100 0 30 0 100 45 0 100 47 1000 55 100 0

Substances

The retention time of compound 3 was typically between 10 and 14 minuteswhen using an HPLC system with a 1.3 minute dwell volume. Compounds 4and 5 co-eluted at longer retention time, with a resolution of ≥2.0.

The relative retention of compound 3 in a sample chromatogram to that inthe standard chromatogram was calculated according to the followingequation:

$R_{r} = \frac{t_{sam}}{t_{std}}$

Where:

-   -   R_(r)=relative retention    -   t_(sam)=retention time of compound 3 peak in the sample        chromatogram, minutes    -   t_(std)=retention time of the drug substance peak in the closest        preceding standard chromatogram, minutes

Assay results were calculated for each sample according to the followingequation:

${\% \mspace{14mu} {assay}} = {\frac{A_{sam}}{A_{std}} \times \frac{W_{std} \times P}{W_{{sam}\;}} \times \frac{1}{\left( \frac{100 - M}{100} \right)} \times 100}$

Where:

-   -   A_(sam)=peak area response of compound 3 in the sample        preparation    -   A_(std)=mean peak area response of compound 3 in the working        standard preparation    -   W_(std)=weight of the standard, mg    -   P=assigned purity of the standard (decimal format)    -   W_(sam)=weight of the sample, mg    -   M=moisture content of the sample, %    -   100=conversion to percent

Relative retention and impurity levels in each sample were calculatedaccording to the following equations:

$R_{r} = \frac{t_{t}}{t_{ds}}$

Where:

-   -   R_(r)=relative retention    -   t_(i)=retention time of the individual impurity    -   t_(ds)=retention time of the compound 3 peak

${\% \mspace{14mu} I_{i}} = {\frac{A_{i} \times W_{std} \times P \times {DF} \times {RF}_{i}}{A_{{std},{1\%}} \times W_{sam}} \times 100}$

Where:

-   -   I_(i)=individual impurity    -   A_(i)=peak area response of individual impurity in the sample        preparation    -   A_(std,1%)=average peak area response of compound 3 in the 1%        standard preparation    -   W_(std)=weight of the standard, mg    -   W_(sam)=weight of sample, mg    -   P=assigned purity of the standard (decimal format)    -   DF=dilution factor, 1/100    -   RF_(i)=relative response factor of individual impurity    -   100=conversion to percentage factor

When assayed by this method,N-(2-pyrazinecarbonyl)-L-phenylalanine-L-leucineboronic anhydride fromExample 2 showed total impurities of less than 1%.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, these particular embodiments areto be considered as illustrative and not restrictive. It will beappreciated by one skilled in the art from a reading of this disclosurethat various changes in form and detail can be made without departingfrom the true scope of the invention and appended claims.

What is claimed is:
 1. A composition comprising an ether solvent thathas low miscibility with water and at least about ten moles of a boronicester compound of formula (I):

wherein: R¹ is an optionally substituted aliphatic or aromatic group; R²is hydrogen, a nucleofugic group, or an optionally substituted aliphaticor aromatic group; R³ is a nucleofugic group or an optionallysubstituted aliphatic or aromatic group; and R⁴ and R⁵ are together anoptionally substituted aliphatic group, and R⁴ and R⁵, taken togetherwith the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring having 0 additional ring heteroatoms;and none of the variables R¹-R⁵ is substituted with a heteroaromaticgroup; wherein the solubility of water in the ether solvent that has lowmiscibility with water is less than about 5% w/w; and wherein the ethersolvent that has low miscibility with water constitutes at least about70% v/v of the reaction mixture.
 2. A composition comprising an ethersolvent that has low miscibility with water and at least about ten molesof a boronic ester compound of formula (I):

wherein: R¹ is an optionally substituted aliphatic or aromatic group; R²is hydrogen, a nucleofugic group, or an optionally substituted aliphaticor aromatic group; R³ is a nucleofugic group or an optionallysubstituted aliphatic or aromatic group; and R⁴ and R⁵ are together anoptionally substituted aliphatic group, and R⁴ and R⁵, taken togetherwith the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring having 0 additional ring heteroatoms;and none of the variables R¹-R⁵ is substituted with a heteroaromaticgroup; wherein the carbon atom to which R¹, R², and R³ are attached is achiral center, having a diastereomeric ratio of at least about 96:4,relative to a chiral center in the R⁴-R⁵ chiral moiety; wherein thesolubility of water in the ether solvent that has low miscibility withwater is less than about 5% w/w; and wherein the ether solvent that haslow miscibility with water constitutes at least about 70% v/v of thereaction mixture.
 3. A composition comprising an ether solvent that haslow miscibility with water and at least about ten moles of a boronicester compound of formula (I):

wherein: R¹ is an optionally substituted aliphatic or aromatic group; R²is hydrogen, a nucleofugic group, or an optionally substituted aliphaticor aromatic group; R³ is a nucleofugic group or an optionallysubstituted aliphatic or aromatic group; and R⁴ and R⁵ are together anoptionally substituted aliphatic group, and R⁴ and R⁵, taken togetherwith the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring having 0 additional ring heteroatoms;and none of the variables R¹-R⁵ is substituted with a heteroaromaticgroup; wherein the carbon atom to which R¹, R², and R³ are attached is achiral center, having an epimeric ratio of at least about 96:4; whereinthe solubility of water in the ether solvent that has low miscibilitywith water is less than about 5% w/w; and wherein the ether solvent thathas low miscibility with water constitutes at least about 70% v/v of thereaction mixture.
 4. The composition of any one of claims 1-3, whereinthe solubility of water in the ether solvent is less than about 2% w/w.5. The composition of any one of claims 1-3, wherein the ether solventis selected from the group consisting of tert-butyl methyl ether,tert-butyl ethyl ether, tert-amyl methyl ether, isopropyl ether, andmixtures thereof.
 6. The composition of any one of claims 1-3, whereinR¹ is C₁₋₈ aliphatic, C₆₋₁₀ aryl, or (C₆₋₁₀ aryl)(C₁₋₆ aliphatic). 7.The composition of any one of claims 1-3, characterized by at least oneof the following features: (a) R³ is chloro; (b) R² is hydrogen; and (c)R¹ is C₁₋₄ aliphatic
 8. The composition of any one of claims 1-3,wherein the compound of formula (I) is


9. A composition comprising at least about ten moles of a boronic estercompound of formula (I):

wherein: R¹ is an optionally substituted aliphatic or aromatic group; R²is hydrogen, a nucleofugic group, or an optionally substituted aliphaticor aromatic group; R³ is a nucleofugic group or an optionallysubstituted aliphatic or aromatic group; and R⁴ and R⁵ are together anoptionally substituted aliphatic group, and R⁴ and R⁵, taken togetherwith the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring having 0 additional ring heteroatoms;and none of the variables R¹-R⁵ is substituted with a heteroaromaticgroup; wherein the carbon atom to which R¹, R², and R³ are attached is achiral center, having a diastereomeric ratio of at least about 96:4,relative to a chiral center in the R⁴-R⁵ chiral moiety; wherein theboronic ester compound of formula (I) constitutes at least about 70% w/wof the composition; wherein the solubility of water in the ether solventthat has low miscibility with water is less than about 5% w/w; andwherein the ether solvent that has low miscibility with waterconstitutes at least about 70% v/v of the reaction mixture.
 10. Thecomposition of claim 3 comprising at least about 20 moles of the boronicester compound of formula (I).
 11. The composition of claim 3, whereinthe carbon atom to which R¹, R², and R³ are attached has adiastereomeric ratio of at least about 97:3, relative to a chiral centerin the R⁴-R⁵ chiral moiety.
 12. The composition of claim 3, wherein allof the boronic ester compound of formula (I) present in the compositionis produced in a single batch run.
 13. The composition of claim 3,wherein at least one of the following features is present: (a) R³ ischloro; (b) the boronic ester compound of formula (I) is:

(c) R² is hydrogen; and (d) R¹ is C₁₋₄ aliphatic.
 14. A large-scaleprocess for preparing a boronic ester compound of formula (I):

wherein: R¹ is an optionally substituted aliphatic or aromatic group; R²is hydrogen, a nucleofugic group, or an optionally substituted aliphaticor aromatic group; R³ is a nucleofugic group or an optionallysubstituted aliphatic or aromatic group; and R⁴ and R⁵ are together anoptionally substituted aliphatic group, and R⁴ and R⁵, taken togetherwith the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring having 0 additional ring heteroatoms;and none of the variables R¹-R⁵ is substituted with a heteroaromaticgroup; said process comprising: (a) providing a solution comprising: (i)a boronic ester of formula (III):

wherein R¹, R⁴, and R⁵ are as defined above; and (ii) an ether solventthat has low miscibility with water, wherein the solubility of water inthe ether solvent that has low miscibility with water is less than about5% w/w; and wherein the ether solvent that has low miscibility withwater constitutes at least about 70% v/v of the reaction mixture; (b)treating the solution with a reagent of formula (IV):

to form a boron “ate” complex of formula (II):

where Y is a nucleofugic group; M⁺ is a cation; and each of R¹ to R⁵ areas defined above; and (c) contacting the boron “ate” complex of formula(II) with a Lewis acid under conditions that afford the boronic estercompound of formula (I), said contacting step being conducted in areaction mixture comprising: (i) a coordinating ether solvent that haslow miscibility with water; or (ii) an ether solvent that has lowmiscibility with water and a coordinating co-solvent, provided that thecoordinating co-solvent constitutes no more than about 20% v/v of thereaction mixture; wherein the solubility of water in the ether solventin (i) or (ii) that has low miscibility with water is less than about 5%w/w; and wherein the ether solvent in (i) or (ii) that has lowmiscibility with water constitutes at least about 70% v/v of thereaction mixture.
 15. A large-scale process for preparing a boronicester compound of formula (I):

wherein: R¹ is an optionally substituted aliphatic or aromatic group; R²is hydrogen, a nucleofugic group, or an optionally substituted aliphaticor aromatic group; R³ is a nucleofugic group or an optionallysubstituted aliphatic or aromatic group; and R⁴ and R⁵ are together anoptionally substituted aliphatic group, and R⁴ and R⁵, taken togetherwith the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring having 0 additional ring heteroatoms;and none of the variables R¹-R⁵ is substituted with a heteroaromaticgroup; said process comprising: (a) providing a solution comprising: (i)a boronic ester of formula (III):

wherein R¹, R⁴, and R⁵ are as defined above; (ii) a compound of formula(V):

where Y is a nucleofugic group, and R² and R³ are as defined above; and(iii) a solvent comprising: (aa) a coordinating ether solvent that haslow miscibility with water; or (bb) an ether solvent that has lowmiscibility with water and a coordinating co-solvent, provided that thecoordinating co-solvent constitutes no more than about 20% v/v of thereaction mixture; wherein the solubility of water in the ether solventin (aa) or (bb) that has low miscibility with water is less than about5% w/w; and wherein the ether solvent in (aa) or (bb) that has lowmiscibility with water constitutes at least about 70% v/v of thereaction mixture; (b) treating the solution of step (a) with a strong,sterically hindered base to form a boron “ate” complex of formula (II):

where M⁺ is a cation derived from the base, and each of Y and R¹ to R⁵are as defined above; and (c) contacting the boron “ate” complex offormula (II) with a Lewis acid in a solution comprising an ether solventthat has low miscibility with water to form the boronic ester compoundof formula (I), wherein the solubility of water in the ether solventthat has low miscibility with water is less than about 5% w/w; andwherein the ether solvent that has low miscibility with waterconstitutes at least about 70% v/v of the reaction mixture.
 16. Alarge-scale process for preparing a boronic ester compound of formula(I):

wherein: R¹ is an optionally substituted aliphatic or aromatic group; R²is hydrogen, a nucleofugic group, or an optionally substituted aliphaticor aromatic group; R³ is a nucleofugic group or an optionallysubstituted aliphatic or aromatic group; and R⁴ and R⁵ are together anoptionally substituted aliphatic group, and R⁴ and R⁵, taken togetherwith the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring having 0 additional ring heteroatoms;and none of the variables R¹-R⁵ is substituted with a heteroaromaticgroup; said process comprising: (a) providing a solution comprising: (i)a boronic acid compound of formula (VI):

wherein R¹ is as defined above; (ii) a compound of formula HO—R⁴-R⁵—OH,wherein R⁴ and R⁵ are as defined above; and (iii) an organic solventthat forms an azeotrope with water; (b) heating the solution of step (a)at reflux, with azeotropic removal of water, to form a boronic ester offormula (III):

wherein R¹, R⁴, and R⁵ are as defined above; (c) providing a solutioncomprising: (i) the boronic ester of formula (III); (ii) a compound offormula (V):

wherein Y is a nucleofugic group, and R² and R³ are as defined above;and (iii) a solvent comprising: (aa) a coordinating ether solvent thathas low miscibility with water; or (bb) an ether solvent that has lowmiscibility with water and a coordinating co-solvent, provided that thecoordinating co-solvent constitutes no more than about 20% v/v of thereaction mixture; wherein the solubility of water in the ether solventin (aa) or (bb) that has low miscibility with water is less than about5% w/w; and wherein the ether solvent in (aa) or (bb) that has lowmiscibility with water constitutes at least about 70% v/v of thereaction mixture; (d) treating the solution from step (c) with a strong,sterically hindered base to form a boron “ate” complex of formula (II):

where M⁺ is a cation derived from the base, and each of Y and R¹ to R⁵are as defined above; and (e) contacting the boron “ate” complex offormula (II) with a Lewis acid in a solution comprising an ether solventthat has low miscibility with water to form the boronic ester compoundof formula (I), wherein the solubility of water in the ether solventthat has low miscibility with water is less than about 5% w/w; andwherein the ether solvent that has low miscibility with waterconstitutes at least about 70% v/v of the reaction mixture.
 17. Theprocess of claim 15 or 16, wherein the sterically hindered base is analkali metal dialkylamide base of formula M²N(R^(#))₂, wherein M² isselected from the group consisting of Li, Na, and K, and each R^(#),independently, is a branched or cyclic C₃₋₆ aliphatic.
 18. The processof claim 16, wherein the organic solvent in step (a) is selected fromthe group consisting of acetonitrile, toluene, hexane, heptane, andmixtures thereof.
 19. The process of claim 16, wherein the organicsolvent in step (a) is an ether solvent that has low miscibility withwater, wherein the solubility of water in the ether solvent that has lowmiscibility with water is less than about 5% w/w.
 20. The process ofclaim 19, wherein the solutions in steps (a) and (c) each comprise thesame ether solvent.
 21. The process of claim 20, wherein step (b)provides a product solution comprising the boronic ester of formula(III), and the product solution from step (b) is used in step (c)without isolation of the boronic ester of formula (III).
 22. Alarge-scale process for preparing an aminoboronic ester compound offormula (VII):

or an acid addition salt thereof, wherein: R¹ is an optionallysubstituted aliphatic or aromatic group; and R⁴ and R⁵ are together anoptionally substituted aliphatic group, and R⁴ and R⁵, taken togetherwith the intervening oxygen and boron atoms, form an optionallysubstituted 5- to 10-membered ring having 0 additional ring heteroatoms;and none of the variables R¹-R⁵ is substituted with a heteroaromaticgroup; said process comprising: (a) providing a boron “ate” complex offormula (II):

where Y is a nucleofugic group; M⁺ is a cation; R² is hydrogen; R³ is anucleofugic group; and each of R¹, R⁴, and R⁵ are as defined above; (b)contacting the boron “ate” complex of formula (II) with a Lewis acidunder conditions that afford the boronic ester compound of formula (I):

where each of R¹ to R⁵ is as defined above, said contacting step beingconducted in a reaction mixture comprising: (i) a coordinating ethersolvent that has low miscibility with water; or (ii) an ether solventthat has low miscibility with water and a coordinating co-solvent,provided that the coordinating co-solvent constitutes no more than about20% v/v of the reaction mixture; wherein the solubility of water in theether solvent in (i) or (ii) that has low miscibility with water is lessthan about 5% w/w; and wherein the ether solvent in (i) or (ii) that haslow miscibility with water constitutes at least about 70% v/v of thereaction mixture; (c) treating the boronic ester compound of formula (I)with a reagent of formula M¹-N(Si(R⁶)₃)₂, where M¹ is an alkali metaland each R⁶ independently is selected from the group consisting ofalkyl, aralkyl, and aryl, where the aryl or aryl portion of the aralkylis optionally substituted, to form a byproduct of formula M¹-R³ and acompound of formula (VIII):

wherein each G is —Si(R⁶)₃ and R¹ to R⁵ are as defined above; and (d)removing the G groups to form a compound of formula (VII):

or an acid addition salt thereof.
 23. The process of claim 22, whereinthe reaction mixture in step (c) comprises an organic solvent in whichthe byproduct M¹-R³ has low solubility.
 24. The process of claim 23,wherein M¹ is Li and R³ is Cl.
 25. The process of claim 24, wherein thereaction mixture in step (c) comprises an organic solvent selected fromthe group consisting of methylcyclohexane, cyclohexane, heptane, hexane,toluene, and mixtures thereof.
 26. The process of claim 22, wherein thereaction in step (c) is conducted at a reaction temperature in the rangeof about −100° C. to about 50° C.
 27. The process of claim 26, whereinthe reaction temperature is in the range of about −50° C. to about 25°C.
 28. The process of claim 26, wherein the reaction temperature is inthe range of about −30° C. to about 0° C.
 29. The process of claim 22,wherein step (d) comprises treating the compound of formula (VIII) withan acid and isolating the compound of formula (VII) as the acid additionsalt.
 30. The process of claim 29, wherein the acid is trifluoroaceticacid.
 31. The process of claim 22, wherein step (c) further comprisesfiltering the reaction mixture to provide a filtrate comprising thecompound of formula (VIII).
 32. The process of claim 31, wherein in step(c), the reagent of formula M¹-N(Si(R⁶)₃)₂ is added to the reactionmixture as a solution comprising tetrahydrofuran, and step (c) furthercomprises removing the tetrahydrofuran before filtering the reactionmixture.
 33. The process of claim 31, wherein the filtrate is useddirectly in step (d).
 34. The process of claim 22, further comprisingthe step: (e) coupling the compound of formula (VII) with a compound offormula (IX):

wherein: P¹ is an amino group blocking moiety; R⁷ is selected from thegroup consisting of hydrogen, C₁₋₁₀aliphatic, optionally substitutedC₆₋₁₀aryl, or C₁₋₆aliphatic-R⁸; and R⁸ is selected from the groupconsisting of alkoxy, alkylthio, optionally substituted aryl,heteroaryl, and heterocyclyl groups, and optionally protected amino,hydroxy, and guanidino groups; and X is OH or a leaving group; to form acompound of formula (X):

wherein each of P¹, R¹, R⁴, R⁵, and R⁷ is as defined above.
 35. Theprocess of claim 34, wherein P¹ is a cleavable protecting group.
 36. Theprocess of claim 35, further comprising the steps: (f) cleaving theprotecting group P¹ to form a compound of formula (XI):

or an acid addition salt thereof, wherein each of R¹, R⁴, R⁵, and R⁷ isas defined above; (g) coupling the compound of formula (XI) with areagent of formula P²—X, wherein P² is an amino group blocking moietyand X is a leaving group, to form a compound of formula (XII):

wherein each of P², R¹, R⁴, R⁵, and R⁷ are as defined above; and (h)deprotecting the boronic acid moiety to form a compound of formula(XIII):

or a boronic acid anhydride thereof, wherein each of P¹, R¹, and R⁷ areas defined above.
 37. A large-scale process for preparing anaminoboronic ester compound of formula (VIIa) or (VIIb):

or an acid addition salt thereof, wherein: R¹ is an optionallysubstituted aliphatic, aromatic, or heteroaromatic group; and R⁴ and R⁵,taken together with the intervening oxygen and boron atoms, form anoptionally substituted chiral cyclic boronic ester; said processcomprising: (a) providing a boron “ate” complex of formula (IIa) or(IIb):

where Y is a nucleofugic group; M⁺ is a cation; R² is hydrogen; R³ is anucleofugic group; and R⁴ and R⁵ are as defined above; (b) contactingthe boron “ate” complex of formula (IIa) or (IIb) with a Lewis acidunder conditions that afford a boronic ester compound of formula (Ia) or(Ib):

where each of R¹ to R⁵ is as defined above, said contacting step beingconducted in a reaction mixture comprising: (i) a coordinating ethersolvent that has low miscibility with water; or (ii) an ether solventthat has low miscibility with water and a coordinating co-solvent,provided that the coordinating co-solvent constitutes no more than about20% v/v of the reaction mixture; wherein the solubility of water in theether solvent in (i) or (ii) that has low miscibility with water is lessthan about 5% w/w; and wherein the ether solvent in (i) or (ii) that haslow miscibility with water constitutes at least about 70% v/v of thereaction mixture; (c) treating the boronic ester compound of formula(Ia) or (Ib) with a reagent of formula M¹-N(G)₂, where M¹ is an alkalimetal and each G is an amino group protecting moiety, to form a compoundof formula (VIIIa) or (VIIIb):

wherein each G and R¹ to R⁵ are as defined above; and (d) removing the Ggroups to form a compound of formula (VIIa) or (VIIb):

or an acid addition salt thereof.
 38. A large-scale process for forminga compound of formula (XIV):

or a boronic acid anhydride thereof, comprising the steps: (aa) couplinga compound of formula (XVIII):

or an acid addition salt thereof, with a compound of formula (XIX):

wherein: P¹ is a cleavable amino group protecting moiety; and X is OH ora leaving group; to form a compound of formula (XX):

wherein P¹ is as defined above, said coupling step (aa) comprising thesteps: (i) coupling the compound of formula (XVIII) with a compound offormula (XIX) wherein X is OH in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) and a tertiary amine in dichloromethane; (ii) performing asolvent exchange to replace dichloromethane with ethyl acetate; and(iii) performing an aqueous wash of the ethyl acetate solution; (bb)removing the protecting group P¹ to form a compound of formula (XXI):

or an acid addition salt thereof, said protecting group removing step(bb) comprising the steps: (i) treating the compound of formula (XX)with HCl in ethyl acetate; (ii) adding heptane to the reaction mixture;and (iii) isolating by crystallization the compound of formula (XXI) asits HCl addition salt; (cc) coupling the compound of formula (XXI) witha reagent of formula (XXII)

wherein X is a OH or a leaving group, to form a compound of formula(XXIII):

said coupling step (cc) comprising the steps: (i) coupling the compoundof formula (XXI) with 2-pyrazinecarboxylic acid in the presence of TBTUand a tertiary amine in dichloromethane; (ii) performing a solventexchange to replace dichloromethane with ethyl acetate; and (iii)performing an aqueous wash of the ethyl acetate solution; and (dd)deprotecting the boronic acid moiety to form the compound of formula(XIV) or a boronic acid anhydride thereof, said deprotecting step (dd)comprising the steps: (i) providing a biphasic mixture comprising thecompound of formula (XXIII), an organic boronic acid acceptor, a loweralkanol, a C₅₋₈ hydrocarbon solvent, and aqueous mineral acid; (ii)stirring the biphasic mixture to afford the compound of formula (XIV);(iii) separating the solvent layers; and (iv) extracting the compound offormula (XIV), or a boronic acid anhydride thereof, into an organicsolvent.
 39. The process of claim 38, wherein step (dd)(iii) comprisesthe steps: (1) separating the solvent layers; (2) adjusting the aqueouslayer to basic pH; (3) washing the aqueous layer with an organicsolvent; and (4) adjusting the aqueous layer to a pH less than about 8.40. The process of claim 39, wherein in step (dd)(iv), the compound offormula (XIV), or a boronic acid anhydride thereof, is extracted intodichloromethane, the solvent is exchanged to ethyl acetate, an thecompound of formula (XIV), or a boronic acid anhydride thereof, iscrystallized by addition of hexane or heptane.
 41. The process of claim40, wherein addition of hexane or heptane results in crystallization ofa cyclic trimeric boronic acid anhydride of formula (XXIV):


42. A large-scale process for forming a compound of formula (XIV): or aboronic acid anhydride thereof. The process comprises the steps:

(a) providing a boron “ate” complex of formula (XV):

wherein: R³ is a nucleofugic group; Y is a nucleofugic group; and M⁺ isan alkali metal; (b) contacting the boron “ate” complex of formula (XV)with a Lewis acid under conditions that afford a boronic ester compoundof formula (XVI):

said contacting step being conducted in a reaction mixture comprising:(i) a coordinating ether solvent that has low miscibility with water; or(ii) an ether solvent that has low miscibility with water and acoordinating co-solvent, provided that the coordinating co-solventconstitutes no more than about 20% v/v of the reaction mixture; whereinthe solubility of water in the ether solvent in (i) or (ii) that has lowmiscibility with water is less than about 5% w/w; and wherein the ethersolvent in (i) or (ii) that has low miscibility with water constitutesat least about 70% v/v of the reaction mixture; (c) treating the boronicester compound of formula (XVI) with a reagent of formulaM¹-N(Si(R⁶)₃)₂, where M¹ is an alkali metal and each R⁶ independently isselected from the group consisting of alkyl, aralkyl, and aryl, wherethe aryl or aryl portion of the aralkyl is optionally substituted, toform a compound of formula (XVII):

wherein each G is —Si(R⁶)₃; (d) removing the (R⁶)₃Si groups to form acompound of formula (XVIII):

or an acid addition salt thereof; (e′) coupling the compound of formula(XVIII) with a compound of formula (XIXa):

wherein X is OH or a leaving group, to form a compound of formula(XXIII):

and (f′) deprotecting the boronic acid moiety to form the compound offormula (XIV) or a boronic acid anhydride thereof.
 43. The process ofclaim 42, characterized by at least one of the following features(1)-(3): (1) In the boron “ate” complex of formula (XV), R³ and Y bothare chloro. (2) The coupling step (e′) comprises the steps: (i) couplingthe compound of formula (XVIII) with a compound of formula (XIXa)wherein X is OH in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) and a tertiary amine in dichloromethane; (ii) performing asolvent exchange to replace dichloromethane with ethyl acetate; and(iii) performing an aqueous wash of the ethyl acetate solution. (3) Theboronic acid deprotecting step (f′) comprises the steps: (i) providing abiphasic mixture comprising the compound of formula (XXIII), an organicboronic acid acceptor, a lower alkanol, a C₅₋₈ hydrocarbon solvent, andaqueous mineral acid; (ii) stirring the biphasic mixture to afford thecompound of formula (XIV); (iii) separating the solvent layers; and (iv)extracting the compound of formula (XIV), or a boronic acid anhydridethereof, into an organic solvent.
 44. The process of claim 43, whereinstep (f′)(iii) comprises the steps: (1) separating the solvent layers;(2) adjusting the aqueous layer to basic pH; (3) washing the aqueouslayer with an organic solvent; and (4) adjusting the aqueous layer to apH less than about
 8. 45. The process of claim 44, wherein in step(f′)(iii)(3), the aqueous layer is washed with dichloromethane.
 46. Theprocess of claim 44, wherein in step (f′)(iv), the compound of formula(XIV), or a boronic acid anhydride thereof, is extracted intodichloromethane, the solvent is exchanged to ethyl acetate, an thecompound of formula (XIV), or a boronic acid anhydride thereof, iscrystallized by addition of hexane or heptane.
 47. The process of claim46, wherein addition of hexane or heptane results in crystallization ofa cyclic trimeric boronic acid anhydride of formula (XXIV):


48. A composition comprising at least one kilogram of a compound offormula (XXIV):

wherein the compound of formula (XXIV) is prepared according to theprocess of claim
 38. 49. A composition comprising at least one kilogramof a compound of formula (XXIV):

wherein the compound of formula (XXIV) constitutes at least 99% w/w ofthe composition.